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1. Geese e Co o te e OC JO OOO i HI POT A a coon JO Oj e NS CJi Communications Cable Power Cable Figure 1 8 Connections of the Applied Biosystems 7900HT Fast Real Time PCR System Table 1 2 Connections of the Applied Biosystems 7900HT Fast Real Time PCR System Cable Type Connects To A Communication Computer Monitor Port Monitor B Comm Power Computer Mouse Port Mouse not shown C Serial Computer Serial Port 1 7900HT Instrument D Comm Power Computer Keyboard Port Hand held Bar Code Reader E Communication Computer Serial Port 2 Plate Handler Port C F Ethernet Network Computer Ethernet Port G Comm Power Computer ISA Card 1 Fixed Position Bar Code Reader H Comm Power Bar Code Reader Cable Keyboard not shown See Figure 1 9 on page 1 11 1 10 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Instrument Connections Fixed Position Bar Code Reader BarCode Reader Cable To Lava Card ISA Card 1 Power supply Power IEC 320 4 Position Universal Power Strip PN 4333969 Universal Voltage Accessory Kit P N 4334482 aa LITT aoe Universal Jumper Cord set x 4 IEC 320 Continental Jumper Cord set for Japan x 4 see
2. Steps 2 and 2 can be eliminated by importing the plate document setup information from a tab delimited text file See Importing Plate Document Setup Table Files on page A 2 for more information Dissociation The SDS software can perform a dissociation curve analysis as part of an absolute Melting Curve quantification run Therefore to perform a dissociation curve construct a plate Workflow document for absolute quantification as explained on page 3 4 and configure the method with a temperature ramp as explained on page 3 21 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 5 Chapter 3 Preparing a Run Allelic Discrimination 1 Create an allelic discrimination plate document see page 3 8 Workflow i 2 Apply markers to the plate document a Create detectors for the allelic discrimination probes See page 3 9 b Create a marker for each allelic discrimination probe pairing see page 3 12 c Copy the marker s to the plate document See page 3 13 3 Assign detector tasks to the wells of the plate document NTC and Unknown see page 3 14 4 lf you would like to perform thermal cycling of the allelic discrimination plate on the 7900HT instrument create a real time plate document for the plate and program it with the method for the allelic discrimination run Otherwise continue to step 2 See page 3 17 5 Choos
3. Figure 6 8 Sample Configuration of the Example Plates Note For more information on the TaqMan Cytokine Gene Expression Plate I see the TaqMan Cytokine Gene Expression Plate I Protocol PN 4306744 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 21 Chapter 6 Analyzing Real Time Data Analysis Checklist Workflow Overview 1 Create an SDS 7900HT Multiple Plate Document See page 6 23 2 Add relative quantification plates for the analysis See page 6 24 3 Configure the analysis settings for the run see page 6 26 4 Analyze the run data see page 6 30 Y 5 For each detector that you configured to determine the baseline and threshold values see page 6 46 Automatically Verify that the baseline and threshold values for the detector were set correctly by the software Manually Set the baseline and threshold values for the detector Y 6 For each detector that you configured for manual outlier removal visualize outliers and eliminate any outlying amplification from the run data see page 6 49 7 View the results of the relative quantification run see page 6 31 v 8 Choose from the following post analysis settings see page 6 35 Reanalyze the run data Adjust the display settings for the results table plate grid and plate document plots Print elements of the plate document
4. 7 Evaluate the filling as follows If all fill reservoirs are Then not uniform after the first centrifuge cycle the Low Density Array may be centrifuged again in the same manner as before for 1 additional min not uniform after the additional centrifuge cycle e The Low Density Array should not be processed further Use another Low Density Array e Alternatively you can process the Low Density Array further however you should void the results for the affected fill reservoir Sample IMPORTANT Do not exceed 1200 rpm or accumulated centrifugation times of more than 3 min Excessive centrifugation speeds and times may deform the Low Density Array 4 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Preparing TaqMan Low Density Arrays for Use Sealing the TaqMan Low Density Arrays Equipment and You need the following equipment and materials to seal the Low Density Arrays Materials Needed Microfluidic card sealer Sturdy lab bench TaqMan Low Density Array s containing sample specific PCR reaction mix and centrifuged page 4 16 Scissors Guidelines for Follow the guidelines below to ensure the Low Density Arrays are properly sealed Sealing the Cards Seal the Low Density Arrays as soon as possible following centrifugation The risk of cross contamination is minimized by the sealing process Always place the Low Density Arrays
5. Fixed position bar code reader cover 2 Power on the computer 3 Start the Automation Controller Software 4 Click open close The 7900HT instrument moves the instrument tray to the out position perpendicular to the instrument 5 Select File gt Exit The software quits the Automation Controller Software Positioning the IMPORTANT The instrument tray must be in the OUT position to align the bar code Fixed Position reader Bar Code Reader 1 Place a plate with bar code onto the instrument tray IMPORTANT Orient the plate so that well A1 aligns to the A1 position of the instrument tray and that the bar code faces the fixed position bar code reader Well position A1 Bar code 2 Select g amp stat gt amp Programs gt amp PSC Laser Data gt LDHOST The LDHOST software starts and displays the LDHOST window Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 49 Chapter 7 Maintaining the Instrument 3 Establish communication with the fixed position bar code reader a Click A Edit b Click Terminal The software opens the Edit Configuration and Terminal dialog boxes c In the Device Control dialog box click Connect to Device The terminal window displays the fixed position bar code reader response d Click to close the Information dialog box The LD Host program communicates with the bar code reader and updates the Edit Configur
6. eyar RE LFA k Edt vew Tods ioi EE g Ej Practice Abe mi Button Tiree Haip 2 3 4151871815 eae e BERR ERR eee BERR REE RR e ee BERR ERE RRR eee 8 te eee tee eg BEER E REE REE BERR REE Ree ee e te eee et eee ee SS SSS SSS F Relative Quantification Multiple Plate Document a Select a plate in the Plates field to view then select all wells of the plate by clicking the upper left corner of the plate grid b Repeat step a to select all wells of every plate in the study Click here to select all wells of the plate grid Select a plate here 3 In the Detector drop down list in the Amplification plot select a detector for which you want to set the baseline and threshold values The software displays the data for the selected wells within the Amplification plot 4 In the Plot drop down list select AR vs Cycle 5 Double click the Amplification plot or click 6 46 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Setting the Baseline and Threshold Values 6 In the Display Settings dialog box a In the Select Pane View field select Amplification Plot b Inthe Y Axis group box of the Scale tab select the Linear option button c Click oo 7 Identify the components of the linear scale Amplification plot and set the baseline so that the amplification curve growth begins at a cycle number greater than the S
7. 30 Wavelength 30 Wavelength E E E E E E FF E A2 AB AA AS AB Ag AB Ag E E E E E E E8 E B fon B2 B3 B4 B5 BG B7 BS B9 E E E E E E E8 E C fc c2 c3 c4 cs c6 c7 cs cg E E E E E E E D fo D2 D3 D4 DS D6 D7 bs bg E E E E E E E E E tes E2 E3 E4 E5 E6 E7 Es E9 E E E E E a F ira F2 F3 F4 F5 F6 F7 Fs Fo E E E E E E E G lot G2 63 64 os G6 G7 68 69 3E 5 E E ae x N x wo x F x a x a E E E E E E E E A a2 a3 Aa AB AB AT As Ag E E E E E E E E B fei B2 B3 B4 B5 B6 B7 B8 89 E E E E E E 8 E Chea c2 c3 ca c5 ce c7 cs co E E E E E E 8 E D fo1 D2 D3 D4 D5 DB D7 Ds pg E E E E E E E E les E2 E3 E4 E5 E6 E7 Es E9 E E E E E E F fr F2 F3 F4 F5 F6 F7 FS F9 E E E E E E E8 E G 62 63 64 65 66 67 68 ca E E E H H2 H3 H4 H5 HB H7 HB Ha E E B10 B11 E E c10 11 E E D10 D11 E E E10 E11 E E F10 F11 A10 A11 E E B10 B11 c10 11 E E D10 D11 E E E10 E11 E E F10 F11 S10 14 A12 B12 C12 D12 E12 F12 G12 H12 8 10 r rature C Zn 20 Wavelength Time s ann Time s nn Time s an 0 0 20 30 Wavelength rature C Time s Ann nn Time s nn a The raw data from the selected wells contains the peak The contaminated well must be in columns 7 12 The raw data from the selected well
8. DECA Sea a EHEHE E E E x E E e A p Rnaser P RNase P RNazeP P RNazeP a E i E E E L __ UNKNSE UN KAS kK UN KAS K UN KAS E cm BHEEEES H J J J B RAW aseP RAW aseP RAW aseP RAW aseP pa A E E E E UNKNSK UN KAS kK UK ASK UN KAS k maeEBEeEEeEs amp TO O enasr RHaseF RHaseF RHaseF RHaszeF r jE E E E E _ UNENSE UK ASK UAW KAS kK UN KAS k om E 5 E z H ID ET pacer M Racer M Racer M Racer 2 Click again to restore the plate grid to the original size 3 Practice zooming portions of the plate grid until you are comfortable using the feature Exercise 4 You can also adjust the size of the plate grid wells by moving the lines in the row or Resizing Wells column headers Using the Border Li Move the mouse cursor over a border line in the row or column header ines The mouse cursor becomes a double arrow gt 2 Click and drag the mouse cursor to adjust the well to a new width The software resizes all wells in the plate grid to match the new width Mouse cursor Ei Practice sds i Practice sds L Ti Fabore a 1 2 3 4 E f 7 A E Fhaser Rna RNase Bj Rhaser OUNKNSK UNKNSK UNKNSK UNKNS5K E E E E B E Razer P RNazerP P RNase P RNazer _ UNENSK UM EMS UM EMS FE UM EMS Ee E a i E l C p Fhar P RNaserP P RNazer P RNase UNKNSK UNENSE UNENSE UNENSE D M RNaser D Anaser MM Rnaser MM
9. 4 32 Operating the Software without an SDS Enterprise Database 4 34 Adding a Plate Document to the Plate Queue Using the SDS Software 4 35 Creating Plate Documents Using the Template Batch Utility 4 36 Starting and Configuring the Automation Controller Software for Operation 4 39 Running Plates Using the Automation Controller Software 4 4 41 Loading Plates onto the Plate Handler 2 000 cece eee eee 4 41 Running the Instrument 0 0 00 ce ee eee 4 43 Ater TAS RUN cack tensa ites lag aa wae ae ate Dee eae ae ede ah ee eee 4 44 Chapter 5 Analyzing End Point Data End Point Runs on the 7900HT Instrument 0 0 00 cee 5 2 Notes for Database Users ore oor hws ars eh ele och Be ee A een We dere aoe el 5 3 Section 5 1 Allelic Discrimination 0 00 ee 5 5 OVEIVICW 6 4 5 deere i aie eat hea iene See aa aE eon na ee tae eet es 5 6 Berne YOUDCOM ob anc atace tos Ore thee eee ee tse ea See cee nee sea ees 5 9 Analysis GMECKNSE aie ieee cise aaa sir ge itn a tnt NOD hie Ne ie taht ide oh aa air ds es 5 10 Opening the Rum Datel sree a a ee es Be ee Re A ee 5 11 Analyzing an Allelic Discrimination RUN eee 5 12 Calling and Scrutinizing Allelic Discrimination Data 000 000 cee aee 5 13 After TNE ANAlVSIS aestas eed eae Gad od me weed a ool ek eat ane week aod tts 5 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Ent
10. Ifthe background plate or Low Density Array is labeled with a bar code click the Barcode field then scan the bar code number using the hand held bar code reader Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 17 Chapter 7 Maintaining the Instrument 5 Click The software creates a plate document with the attributes for a background run IMPORTANT Do not modify the background plate document The method for a Background run is coded into the SDS software and consists of a single hold at 60 C for 2 min Because the plate contains only deionized water the plate document does not require sample or detector labels 6 Save a b C the plate document Click or select File gt Save In the File name field of the Save dialog box enter Background_ lt date in MMDDYY format gt For example the file name for a background plate run on May 31 2001 would be Background_053101 Click __sve_ The software saves the plate document and is now configured for the Background run 7 Continue with Running the Prepared Background Plate or TaqMan Low Density Array below Running the Prepared Background Plate or TaqMan Low Density Array Performing the 1 Load the background plate into the 7900HT instrument Background Run 7 b C In the plate document in the SDS software select the Instrument tab In the lower portion tab select the Real Time
11. Allele Calls a In the plate grid select the wells containing the No Template Control samples The software highlights the datapoints within the allele plot b Check that the datapoints cluster in the expected position on the plot c If using positive controls repeat steps a and b for the wells containing the Allele X and Allele Y controls Setup Instrument Results Marker Gea Call a FANANA arker a Pk elna Allele Y controls Po aii eit fF 13 Legend Allele X Allele Y Allele X amp Y E NTC X Undetermined Allele CYP 2C19 2 1 Allele X controls should cluster here No Template 1 l l l Controls should Allele X CYP 2C19 2 2 cluster here 2 Designate samples that did not cluster tightly as Undetermined Samples that did not cluster tightly may e Contain rare sequence variations e Contain sequence duplications e Contain contaminants e Bethe result of errors in pipetting or sample preparation Setup Instrument Results Marker I can MAANA Legend Allele X Allele Y Allele X amp Y E NTC X Undetermined Allele Y CYP 2C19 2 1 Undetermined samples Allele X CYP 2C19 2 2 5 16 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Calling and Scrutinizing Allelic Discrimination Data 3 Screen for Unknown samples that failed to amplify a Inthe Allelic Discrimination Plot
12. Currently the SDS software supports one type of end point analysis Allelic Discrimination Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Notes for Database Users Notes for Database Users Database Alerts and Notifications Reason s For Change Dialog Box Electronic Signature Verification Dialog Box If you are using an SDS Enterprise Database to store SDS plate documents sessions studies and data you may be required to perform additional tasks when analyzing Allelic Discrimination data as described in this chapter This section describes the types of actions you may need to perform depending on how your administrator has configured the database For more information on any of the features described below see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide The Reason s for Change dialog box appears only if the SDS Enterprise Database is configured for auditing The auditing system monitors the creation modification and deletion of the SDS data contained by the database When the Reasons for Change dialog box appears do one of the following e Select a description for the change from the drop down list then click ox e Enter a description of the reason for the change in the field then click Reasons For Changa Reasons for change drop down list Resson For Change Atirbuts 4
13. Help button Position Sample Name Marker Hame y Al HO1 1 CYP 209 2 Allele g AT HM1 1 OYP Irar Allala Exporting Plots as Graphics The SDS software can export most panes and plots of the plate document as JPEG Joint Photographic Experts Group graphic files The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software To export a plot as a graphic file see Exporting Graphics on page A 16 or click m within the plot of interest for further instructions 5 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide After the Analysis Saving the The SDS software saves the results of the analysis differently depending on whether Results the analysis is saved to the SDS Enterprise Database see below or to a plate document file see page 5 20 Saving the Results to the SDS Enterprise Database If you use the SDS Enterprise Database you can save your analysis of the run data to the database as an analysis session IMPORTANT Observe the following when saving sessions to the database e If you modified the plate document information in any way by removing wells from use or by changing the display settings you must save the plate document to the database for the changes to persist select File gt Save Plate Document to Database e The SDS Enterpri
14. Replace the cover for the fixed position bar code reader from step 1 on page 7 49 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 51 Chapter 7 Maintaining the Instrument Cleaning and Replacing Gripper Finger Pads When to Perform Materials Required Cleaning the Finger Pads Replacing the Finger Pads The adhesive used to affix bar code labels to certain brands of microplates can build up on the gripper pads of the Zymark Twister Microplate Handler Over time the residue can cause the gripper pads to stick to the microplates while handling them causing misfeeds To prevent buildup inspect the gripper pads monthly and clean or replace the pads as needed Table 7 3 Materials Required for Replacing the Gripper Finger Pads Material Part Number Finger Pad Replacement Kit containing 10 finger pads 4315472 Flat blade screwdriver small Phillips head screwdriver small lsopropanol in a squeeze bottle Nee CHEMICAL HAZARD Isopropanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin and cause irritation It may cause central nervous system effects such as drowsiness dizziness and headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To clean the finger pads wipe e
15. XXIV Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Product Overview In This Chapter System Overview 4c 6iccse besensednss duce baondssadacinweducuawd sos 1 2 Section 1 1 Getting to Know the Hardware c ccc eeecccccccees 1 3 Tas A 3k 694 5 0 6k ee do bak r 940s 1 4 Rs po oo ea EE EN ES eG ee EEE E ea ees 1 6 eg Be 2 a ee ee ee re eee oe eee eee eee ee eee ee 1 7 Zymark Twister Microplate Handler 0 0 0 1 8 Compatible Consumables s o465 itd stun eveeee tesee ved Irr r EEr Ekia 1 9 instrament ONCE uo oes 44 aa R READE ORE R 1 10 Section 1 2 Getting to Know the Software ccc cece cece eee ees 1 13 ae Cec oh oe he 4b ee beh eh ban ee eee ee boas es 1 14 SDS Software Related Files and Formats 0 0 00 cc cece eee eee 1 15 Managing Sequence Detection System Data 0 0 00 0 cece eee 1 17 Section 1 3 SDS Enterprise Database ccc ccc eee cece ees 1 21 About the SDS Enterprise Database Feature 00 00 0000 cea 1 22 About the SDS Enterprise Database Software Suite 0004 1 25 Database Design and Information Management 00000 00 1 27 Supporting API Documentation 44 444 0 4464 420i 0 eRe e eee ade ORS VeRO eS 1 28 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 1 Chapter 1 Product Overview System Overview About the 7900HT Instrument Supported Run
16. e Well number The first tab delimited text block defines the number of the well on the plate Well numbers start at 1 for well A 1 upper left corner of the plate and increases from left to right and from top to bottom The wells must be listed in order 1 2 3 e Sample name The second text block defines the name of the sample assigned to the well e Marker assignments The remaining tab delimited text blocks for the well definition define the marker assigned to the well Each marker is represented by two text blocks that define the following information The name of the marker The task assignment of the marker for the well UNKN Unknown NTC No Template Control To assign more than one marker to a well repeat the marker definition text blocks for each detector There is no limit to the number of markers that can appear in a well IMPORTANT All markers that appear in this section must have been previously defined in the Marker Definitions section elements 7 9 A 12 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Setup Table Files Format for a single well Example Example Code A 12 Allelic discrimination setup table files Well number lt tab gt SDS Sample Name lt tab gt Marker name lt tab gt Marker task lt tab gt Marker name lt tab gt Marker task lt tab gt lt cr gt SDS Setup File Version 3 Output Plat
17. IMPORTANT If a well is removed from use before the plate document is run the SDS software does not collect data for the well during the run However if a well is removed from use after the plate document has been run the software excludes the well from the analysis but does not delete the data 1 If omitting wells from a Relative Quantification Multiple Plate Document select a plate in the Plates field that contains wells you want to omit 2 While pressing and holding the Ctrl key select the well s in the plate grid that you want to omit 3 If omitting wells from a Relative Quantification Multiple Plate Document repeat steps through 2 for each plate in the study 4 Select the Setup tab 5 In the well inspector click the Omit Well s check box to exclude the selected wells from the analysis ald x Selected wells removed from use we z Bests SEES ee ap Omit Well s check box selected rron Note To reinstate an omitted well select the well then click the Omit Well s check box 6 Click or select Analysis gt Analyze 7 Repeat the procedures in Visualizing Outliers on page 6 49 to verify that all outliers have been removed from the analysis 6 50 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide After the Analysis After the Analysis User Access Requirement Post Analysis Options Changing the Disp
18. Kits Reagents and Consumables for a list of compatible consumables and reagents When amplifying samples that contain very low quantities of nucleic acid generally less than 100 molecules expect lowered precision due to the Poisson distribution and biochemical effects related to binding probabilities Low copy templates are also more susceptible to losses due to non specific adhesion to plastic wells pipettor tips etc The addition of carrier to the sample such as yeast tRNA or glycogen can help prevent these losses increasing the precision and sensitivity of the assay The Applied Biosystems buffer contains an internal passive reference molecule ROX dye which acts as a normalization factor for fluorescent emissions detected in the samples IMPORTANT Non Applied Biosystems PCR buffers may not contain the ROX passive reference dye If running non Applied Biosystems chemistry be sure to set the passive reference for your experiment as explained on Setting the Passive Reference on page 3 16 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Background Runs Background Runs Background Table 8 2 Troubleshooting Background Runs Troubleshooting Table Observation Possible Cause Recommended Action Software will not extract During setup the wrong Run a new background background data plate type was assigned to plate document with the the plate document proper plate type
19. e Do not remove safety labels or disable safety interlocks Refer to the user documentation provided with the laser for additional information on government and industry safety regulations ANIA LASER HAZARD Lasers can burn the retina causing permanent blind spots Never look directly into the laser beam Remove jewelry and other items that can reflect the beam into your eyes Do not remove the instrument top or front panels Wear proper eye protection and post a laser warning sign at the entrance to the laboratory if the top or front panels are removed for service ASI LASER BURN HAZARD An overheated laser can cause severe burns if it comes in contact with the skin DO NOT operate the laser when it cannot be cooled by its cooling fan Always wear appropriate laser safety goggles Bar Code Scanner Laser Safety Laser Classification The bar code scanner included with the Applied Biosystems 7900HT Fast Real Time PCR System is categorized as a Class 2 II laser Laser Safety Class 2 II lasers are low power visible light lasers that can damage the eyes Requirements Never look directly into the laser beam The scanner is designed to prevent human access to harmful levels of laser light during normal operation user maintenance or during prescribed service operations ASI LASER HAZARD Class 2 II lasers can cause damage to eyes Avoid looking into a Class 2 II laser beam or pointing a Class 2 II laser beam into anothe
20. 1 Click and drag the grey line dividing the plate grid and well inspector to the right The software expands the plate grid and table pane to the new width 2 Using the grey dividing lines resize other elements of the plate document until you are comfortable using the feature Exercise 2 Hiding You can also toggle the presence of the plate document panes views and plots using and Showing the icons in the Display toolbar Panes Views 1 In the Display toolbar click H the Hide Show Table Pane button and Plots The software removes the table pane from the plate document 2 Click again to show the table pane The software restores the table pane to the plate document The display toolbar can be particularly useful for manipulating information shown in the plate document See About the Display Toolbar on page 2 8 for a list of the other icons of the display toolbar and the panes they control 3 Practice hiding and showing the other plate document elements by clicking other buttons in the Display toolbar until you are comfortable using the feature Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 21 Chapter 2 Getting Started Exercise 3 You can maximize the panes views and plots of plate documents by clicking the Maximizing sizing buttons embedded inside the grey dividing lines Minimizing Note Sizing buttons are the arrowhead marks G that appear between adjacent P
21. 382 P22 383 P23 384 P24 Information for wells 11 through 381 has been removed from this example for brevity 384 NU03000081 8 Quencher Description 4 AlleleY Description AL1 1 AL1 4 AL1 3 AL1 2 Detector Task Marker Task SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP1 UNKN SNP4 UNKN SNP4 UNKN SNP4 UNKN Comments Comments Quantity Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 15 Appendix A Software Reference Exporting Graphics Exporting a Plot The SDS software can export most panes and plots of the plate document as JPEG as a JPEG Joint Photographic Experts Group graphic files The JPEG file format is Graphic File compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software To export an element of a plate document as a graphic l 2 Select the plot or grid you want to export Choose from the following If exporting a plot adjust its dimensions length and width as you want them to appear in the exported graphic file The exported graphic file retains the dimensions of the original screen element If exporting the plate grid do not adjust the size of the wells The software captures the whole grid regardless of the size of the view Right cl
22. Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Applied Biosystems Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Applie App Sems AS Copyright 2007 2010 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures The Applied Biosystems 7900HT Fast Real Time PCR System is a real time thermal cycler covered by US patents and corresponding claims in their non US coun terparts owned by Applied Biosystems No right is conveyed expressly by implication or by estoppel under any other patent claim such as claims to apparatus reagents kits or methods such as 5 nuclease methods Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA TRADEMARKS ABI PRISM Applied Biosystems MicroAmp Primer Express and VIC are registered trademarks and AB Design ABI PRISM Applera FAM JOE NED ROX TAMRA and TET are trademarks of Applied Biosystems or its subsidiaries in the US and or certain other countries AmpErase AmpliTaq Gold GeneAmp and TaqMan are registered trademarks of Roche Molecular Systems Inc SYBR is a registered trademark of Molecular Probes Inc Zymark is a registered trademark of Zymark Corporation Windows and Windows NT are registered trademarks o
23. Replace the plate inside the gripper and listen for the switch to engage f you do not hear the switch engage then remove the plate and repeat steps 1 and 2 above If you hear the switch engage remove the plate and continue to step a below Move the switch Up by turning the thumb wheel one step in the direction indicated on the arm Replace the plate and listen for the switch to engage If you hear the switch engage remove the plate and repeat steps 1 and 2 above If you do not hear the switch engage then you have successfully identified the zero point of the plate sensor switch Note At the zero point one step of the thumb wheel in the Down direction causes the switch to engage 5 After the zero point is established carefully turn the thumb wheel in the Down direction the number of steps appropriate for your plate format as indicated below Consumable Turn the thumb wheel in the Down direction ABI PRISM 96 Well Optical 20 steps Reaction Plate ABI PRISM 384 Well Optical 15 steps Reaction Plate TaqMan Low Density Array 15 steps Note If you lose count begin again from step 4 and identify the zero point for the switch 6 Test the adjustment as explained on page 7 39 7 38 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Testing the 1 Adjustment Adjusting the Sensitivity of the Plate Sensor Switch Place the re
24. Swinkels D W Use of real time quantitative PCR to compare DNA isolation Reagents and methods Clin Chem 44 10 2201 2204 1998 Nucleic Acid Goldsworthy S M Stockton P S Trempus C S Foley J F and Maronpot R R Preparations Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue Mol Carcinog 25 2 86 91 1999 Kruse N Pette M Toyka K and Rieckmann P Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood J Immunol Methods 210 2 195 203 1997 Lie Y S and C J Petropoulos Advances in quantitative PCR technology 5 nuclease assays Curr Opin Biotechnol 9 1998 43 48 Orlando C P Pinzani and M Pazzagli Developments in quantitative PCR Clin Chem Lab Med 36 1998 255 269 Smetsers T F Stevens E H van de Locht L T and Mensink E J Freezing of PCR master mixture retains full amplification activity and facilitates PCR standardisation for molecular diagnostics and real time quantitative PCR letter Leukemia 12 8 1324 1325 1998 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Bibliography 1 General Papers Arnold B A Hepler R W and Keller P M One Step Fluorescent Probe on Absolute Product Enhanced Reverse Transcriptase Assay BioTechniques 25 1 98 106 1998 Quantitation Ambs S S Dennis J Fairman M Wright and J Papkoff 1999 Inhibition of tumor
25. Table C 3 Consumables for Instrument Maintenance and Verification Part Number Description Sequence Detection Systems Spectral Calibration Kits Quantity 4328639 ABI PRISM 7900HT Sequence Detection Systems 96 Well Spectral Calibration Kit Includes three ABI PRiSm 96 Well Optical Reaction Plates one preloaded and sealed background plate and two preloaded and sealed Spectral Calibration plates containing eight separate dye standards FAM JOE NED ROX SYBR Green TAMRA TET VIC 3 x 96 Well Plates 4323977 Sequence Detection Systems 384 Well Spectral Calibration Kit Includes two ABI PRiSM 384 Well Optical Reaction Plates one preloaded and sealed background plate and one preloaded and sealed Spectral Calibration plate containing eight separate dye standards FAM JOE NED ROX SYBR Green TAMRA TET VIC 2x 384 Well Plates 4351653 7900HT System Fast 96 Well Spectral Calibration Kit Includes three Optical 96 Well Fast Thermal Cycling Plates one preloaded and sealed background plate and two preloaded and sealed pure dye plates containing eight separate dye standards FAM JOE NED ROX SYBR Green TAMRA TET VIC TaqMan RNase P Instrument Verification Plates 3 x 96 Well Plates 4310982 TaqMan RNase P Instrument Verification Plate Includes one ABI PRISM 96 Well Optical Reaction Plate Each well contains preloaded re
26. or O N To save the run data to the SDS 7900HT Document 1 In the SDS software select File gt Save To save the run data to the SDS Enterprise Database IMPORTANT If you saved the plate document to the database prior to running the plate the SDS software stored the run data as a temporary file in the temp directory If you closed your plate document at the end of the run without saving it open the temp directory and rename the temporary file to sds and you will be able to open the file 1 Select File gt Save Document to Database 2 In the Comment field of the Save Document to Database dialog box enter a brief description of the plate document up to 255 characters Note If saving a plate document file to the database you will loose the file name unless you enter it into the Comments field Click 4 Inthe Saved Document dialog box click Analyzing the Section 5 1 Allelic Discrimination 0000 eee eee 5 5 Run Data Section 6 1 Absolute Quantification 0 0 00 e eee eee eee eens 6 5 Section 6 2 Relative Quantification sins dose ns e 58 Oh 4 0w 9 9844594 FEdS oS 6 15 Section 6 3 Dissociation Curve Analysis 0 0 eee eens 6 37 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 29 Chapter 4 Operating the Instrument Disconnecting The SDS software has the ability to halt all communications with the 7900HT the Software
27. 0 nnna anaana aaaea Comparative Cy Method of Relative Quantification 004 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Appendix D Theory of Operation Fluorescent Based Chemistries Fundamentals of the 5 Nuclease Assay The PCR reaction exploits the 5 nuclease activity of AmpliTaq Gold DNA Polymerase to cleave a TaqMan probe during PCR The TaqMan probe contains a reporter dye at the 5 end of the probe and a quencher dye at the 3 end of the probe During the reaction cleavage of the probe separates the reporter dye and the quencher dye which results in increased fluorescence of the reporter Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye Figure D 1 shows the forklike structure dependent polymerization associated 5 3 nuclease activity of AmpliTaq Gold DNA Polymerase during PCR Polymerization R Reporter Q Quencher ee CERIA g O y 5 3 Reverse Primer Strand displacement 5 3 5 5 Cleavage R S a ou Ol Polymerization completed R _ N Probe 3 4 5 3 5 5 3 5 Figure D 1 5 3 Nuclease Activity of AmpliTaq Gold DNA Polymerase When the probe is intact the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Forster type energy transfer Forster 1948 Lakow
28. 0 0 0 0 ccc eens C 3 Instrument Maintenance and Verification 0 0 cee eee eee C 5 TaqMan Pre Developed Assays and Reagents 00 0c eee eee C 6 Custom Oligonucleotide Synthesis e465 coos 644 ok 4444s 09s 1k e eS C 6 Note Part numbers listed in this appendix are for customers inside the United States Contact your Regional Sales Office for local Part numbers and prices See How to Obtain Services and Support on page xii Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide C 1 Appendix C Kits Reagents and Consumables Interchangeable Sample Block Modules and Accessories The 7900HT instrument features a Peltier based interchangeable sample block module based on the technology established in the GeneAmp PCR System 9700 thermal cycler The use of an interchangeable sample block module e Reduces instrument downtime by allowing immediate replacement of the block e Permits easy access to the sample block for troubleshooting and maintenance see page 7 14 e Supports multiple consumable formats e Provides several different modes of operation including Max mode and programmable temperature ramps Table C 1 Sample Block Modules and Accessories Part No Description Quantity 4331406 7900HT System Standard 384 Well Block Upgrade Kit 1 kit Includes a Standard 384 Well Block a 384 well plate adapter and a Sequence Detection Systems 384 Well Spec
29. BlankTemplate sisi Browse Barcode Practice E conce 4 Click The software displays a new plate document with appropriate attributes 5 Open the plate document template for the TaqMan RNase P Instrument Verification Plate PN 4310982 for the Standard 96 Well Block and export its plate setup as explained on page 2 14 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 13 Chapter 2 Getting Started Exercise 2 As an alternative to setting up plate documents manually as described in Chapter 4 Exporting Data Operating the Instrument the SDS software can import plate document setup from a Plate information directly from tab delimited Assay Plate Setup Files You can export Document Assay Plate Setup Files from the SDS software or create them using a third party application such as spreadsheet or LIMS software see Appendix A Software Reference for more information In the following exercise you will export the Assay Plate Setup File for a plate document template so that you will import in Exercise 3 Importing Setup Table Data into a Plate Document on page 2 16 Note In addition to Assay Plate Setup Files the SDS software can also export data from most of the analysis plots graphs and tables See Appendix A Software Reference for more information Opening the Plate Document Template In the following procedure you will open the plate documen
30. Desktop 7900HT The software runs each plate Computer Instrument using the method stored in the downloaded plate document During the run the software saves the raw data to the plate document in the database The RQ Manager SNP DaranaSE Manager or SDS software Serer e Studi is used to download and ee oe Immediately after each run the software performs a primary analysis analyze analysis sessions or studies NS multicomponenting and normalization of the raw data The software then saves the analyzed data to the database as an analysis session and attaches it to the appropriate study i e Run Data e Analysis Sessions Figure 1 18 SDS Enterprise Database Data Flow Diagram Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 27 Chapter 1 Product Overview Types of Retrievable Data Persisted Plate Documents Persisted plate documents include the equivalent information stored in SDS plate documents Plate documents have a 1 1 correspondence with the optical plate or Low Density Array they represent Each persisted plate documents consists of a collection of plate setup information for running a plate on the 7900HT instrument including
31. Export the plate document results table or plots 6 22 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Options for Analyzing Relative Quantification Data Options for Analyzing Relative Quantification Data SDS Multiple Plate Documents for Small Scale Analyses RQ Manager Software for Medium to Large Scale Analyses Applied Biosystems provides two ways to analyze data collected for relative quantification experiments In the case of relative quantification the SDS software uses a different kind of plate document called an SDS 7900HT Multiple Plate Document sdm to analyze data from small relative quantification studies consisting of less than 10 plates Unlike file based plate documents multiple plate documents save the settings and results of an analysis Regardless of whether you are analyzing data from a single plate or from multiple plates in a series you must create a multiple plate document to conduct the analysis Note The SDS software does allow you to open individual relative quantification plate documents however they cannot be used individually to generate gene expression values The RQ Manager Software is a stand alone application used to conduct medium to large scale analyses of relative quantification gene expression data stored on an SDS Enterprise Database The softwar
32. Handler must be powered off at all times during the following procedure Failure to comply can result in physical injury to the user or damage to the Plate Handler 2 Clear the switch position by turning the thumb wheel all the way to the Up extreme as indicated on the side panel Thumb wheel Plate sensor switch 3 Begin the adjustment of the sensor switch a Grasp an optical plate by the sides making sure not to place pressure in the center of the plate to deform it b Place the plate between the fingers of the gripper assembly and align it to the middle of the centering device c While holding the plate in position slowly turn the thumb wheel to lower the switch onto the reaction plate until the switch Contacts the top of the plate and Emits a soft audible clicking noise IMPORTANT The sound emitted by the sensor switch is very faint and may be difficult to hear To make the adjustment easier place your ear close to the sensor switch while making the adjustment and listen for the switch to engage Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 37 Chapter 7 Maintaining the Instrument 4 Remove the plate and listen for the plate sensor switch to disengage Did you hear the switch disengage Then No Ja 2 Yes 1 2 Move the switch Down a few steps by turning the thumb wheel in the direction indicated on the arm
33. Hofmann Lehmann and H Lutz 1999 Quantitative real time PCR for the measurement of feline cytokine mRNA Vet Immunol Immunopathol 71 291 305 Maeda I Takano T Matsuzuka F Maruyama T Higashiyama T Liu G Kuma K and Amino N Rapid screening of specific changes in mRNA in thyroid carcinomas by sequence specific differential display decreased expression of acid ceramidase mRNA in malignant and benign thyroid tumors In Process Citation Int J Cancer 81 5 700 704 1999 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Bibliography 5 Mandigers C M Meijerink J P Raemaekers J M Schattenberg A V and Mensink E J Graft versus lymphoma effect of donor leucocyte infusion shown by real time quantitative PCR analysis of t 14 18 letter Lancet 352 9139 1522 1523 1998 Marcucci G et al Detection of minimal residual disease in patients with AML1 ETO associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay Leukemia 12 1998 1482 1489 Mensink E et al Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real time quantitative RT PCR Br J Haematol 102 1998 768 774 Moody A Sellers S and Bumstead N Measuring infectious bursal disease virus RNA in blood by multiplex real time quantitative RT PCR Virol Methods 2000 Mar 85
34. In the Export dialog box select Export gt Setup Table Select the All Wells radio button Select the SDS 2 2 1 radio button In the File name field enter Practice 5 cE Look in _ Templates Fecent Export Setup Table ie From o o Allele Selected ells sps22 C sps24 E Group by Replicates RUE Faol a File name Practice Export Network Files of type Tab delimited Tet Ptt Cancel Click Beet The software saves the plate document setup table information to a tab delimited text file entitled Practice txt Note If desired you can open the Practice txt Assay Plate Setup File using the Microsoft Notepad Wordpad or Word software to view its contents Select File gt Close to close the plate document template IMPORTANT Do not close the plate document created in Exercise 1 Creating a Plate Document on page 2 13 Import the exported plate setup information into the plate document as explained on page 2 16 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 15 Chapter 2 Getting Started Exercise 3 Asa timesaving device the SDS software allows you to import plate document setup Importing Setup information from exported or fabricated Assay Plate Setup Files To illustrate this Table Data into a feature import the plate grid setup information contained in the Practice txt file Plate Document from Exercise 2
35. Indicates a potentially hazardous situation that if not avoided could result in death or serious injury N py leiaag Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTS each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard icons that are affixed to Applied Biosystems instruments see Safety Symbols on page xv Examples The following examples show the use of safety alert words IMPORTANT You must create a separate a Sample Entry Spreadsheet for each 96 well microtiter plate AN o7 The lamp is extremely hot Do not touch the lamp until it has cooled to room temperature Nem CHEMICAL HAZARD Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves JN DANNE ELECTRICAL HAZARD Failure to ground the instrument properly can lead to an electrical shock Ground the instrument according to the provided instructions xiv Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Symbols on Instruments Symbols on Instruments Electrical The
36. Longer runs and runs configured to collect data at multiple stages of the method can be considerably larger than the 15 25 MB average 5 Click the Sample Volume uL field and enter the volume of the reactions on the plate Note Sample volume refers to the entire contents of any well including buffer blank or any combination of master mix and nucleic acids IMPORTANT All wells on one plate must contain the same reaction volume 6 Choose from the following options If performing an Then absolute or relative go to Step 6 Saving the Plate Document as a quantification run only Template on page 3 22 absolute quantification run add a temperature ramp to generate dissociation with a dissociation curve curve data as explained on page 3 21 3 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Step 5 Programming the Plate Document Method Programming a To generate the data required to perform a dissociation curve analysis the 7900HT Temperature instrument must be programmed to run a temperature ramp in which it slowly Ramp for elevates the temperature of the samples while collecting fluorescence measurements Dissociation once every 7 10 seconds see page 6 38 for a detailed explanation Curve Analysis 4 2 3 In the Instrument tab of the plate document select the Thermal Profile tab Click the step to the left of the location you want to place
37. Undetermined 30 Endo Max Max not displayed Target Max Max Undetermined Endo Max not displayed 30 Target Max Max Undetermined Endo lt Max not displayed 30 Target lt lt Undetermined 30 20 Endo Max Max not displayed Target lt Max Undetermined 35 Endo Max lt not displayed 33 Target lt Max Undetermined 30 Endo lt Max not displayed 20 Target Max lt Undetermined 30 Endo Max lt not displayed 20 Target Max Max Undetermined Endo z lt not displayed 20 20 Target Max lt Undetermined 35 Endo lt Max not displayed 33 Target lt Undetermined 30 33 Endo Max not displayed 35 Target lt lt Undetermined 33 30 Endo Max lt not displayed 35 a lt Indicates that the sample C is less than the maximum PCR cycle of the run a valid C7 bMax or Indicates that the amplification curves for the associated sample replicate group never crossed the threshold during the duration of the run undetermined values 6 32 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Viewing Results About Down _ The software displays a down arrow to indicate that a level of expression represents Arrows the maximum target gene expression level The software displays the down arrow on a sample bar when e The calibrator sample for the detector group yields va
38. Using SDS Plate Documents Well Inspector The Well Inspector Figure 2 9 applies detector and sample information to the wells inside the grid pane and displays information from the selected cells in the plate grid Setup Instrument F Sample Name i Sample name field Use Detector Reporter Task Quantity Color a O RNaseF FAN Mixed 0 Detector list Add Detector Passive Reference ROX Passive Reference drop down list T Omit Wells Figure 2 9 Components of the Well Inspector Omit Well check box e Sample Name text field An editable field that displays the sample name applied to the selected well s Note The Sample Name field will display Mixed if multiple wells with different sample names are selected e Detector list Lists all available detectors copied to the plate document e Omit Well check box Toggles the activity of the well If selected the software eliminates the data from the selected well from all analysis procedures e Passive Reference drop down list Displays the fluorescent dye used as a passive reference Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 31 Chapter 2 Getting Started 2 32 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Preparing a Run In This Chapter Noes Tor Database LIGN ni4kb 45sec eee se bead Ob4ekesadeeiouhbesnsan esd 3 2 De SOC no
39. b In the Y Axis group box of the Scale tab select the Log option button c Click 10 Identify the components of the amplification curve and set the threshold so that it is e Above the background e Below the plateaued and linear regions of the amplification curve e Within the geometric phase of the amplification curve If you are inspecting an AutoCalled detector verify that the threshold is called correctly by the software and proceed to step 11 If the threshold is set incorrectly open the Analysis Settings dialog box configure the detector for manual calling then click After the software adjusts the data manually set the baseline and threshold for the detector as described in this procedure 2 000 E 1 000 E 1 Plateau phase Linear phase Geometric phase Threshold setting click and drag 2 166 E1 Background D 24 6 8 10 12 14 fio 18 20 22 24 26 28 30 32 34 36 38 4 L Baseline Cycle 11 Repeat steps 3 through 10 to set the baseline and threshold values for all remaining detectors present on the plate 12 If you choose to eliminated outliers e Automatically View the results e Manually Visualize and eliminate outliers from the analyzed run data as explained in Eliminating Outlying Amplification on page 6 49 6 48 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Eliminating Outlying Amplification Eliminating Outlying Ampl
40. growth correlates with the expression level of a human angiostatin transgene in transfected B16F10 melanoma cells In Process Citation Cancer Res 59 5773 5777 1999 Becker K D Pan and C B Whitley 1999 Real time quantitative polymerase chain reaction to assess gene transfer Hum Gene Ther 10 2559 2566 1999 Berg T M ller A R Platz K P Hohne M Bechstein W O Hopf U Wiedenmann B Neuhaus P and Schreier E Dynamics of GB virus C viremia early after orthotopic liver transplantation indicates extrahepatic tissues as the predominant site of GB virus C replication Hepatology 29 1 245 249 1999 Chiang P W Wei W L Gibson K Bodmer R and Kurnit D M A fluorescent quantitative PCR approach to map gene deletions in the Drosophila genome In Process Citation Genetics 153 3 1313 1316 1999 de Kok J B Hendriks J C van Solinge W W Willems H L Mensink E J and Swinkels D W Use of real time quantitative PCR to compare DNA isolation methods Clin Chem 44 10 2201 2204 1998 Fairman J Roche L Pieslak I Lay M Corson S Fox E Luong C Koe G Lemos B Grove R Fradkin L and Vernachio J Quantitative RT PCR to evaluate in vivo expression of multiple transgenes using a common intron In Process Citation BioTechniques 27 3 566 70 572 4 1999 Gerard C J et al Improved quantitation of minimal residual disease in multiple myeloma using real time polymerase chai
41. verity default is 1521 Service Name field Enter the name of the database default is SDS22 6 Test the new connection a Click veiy to test the new connection The software briefly checks that a database is at the given IP address If the connection is successful the software displays the Login dialog box b In the Test Login dialog box enter your User Name and Password then click User Mare Password e Ifthe SDS software is unable to successfully connect to the database the software displays the error message shown below 7 In the Options dialog box click 8 Log into the database by entering your user name and password then click Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 23 Appendix A Software Reference A 24 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Designing TaqMan Reagent Based R Assays In This Appendix Assay Development Guidelines onnan naana nunn Design Tips for Allelic Discrimination AssaysS 0 0 00 cc eee eee Design Tips for Quantitative PCR AssaysS 0 0 0 0 eee ees Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Appendix B Designing TaqMan Reagent Based Assays Assay Development Guidelines TaqMan Reagent l Based Assay Development Program a es a ee oe Identify target sequence
42. 1 2 55 64 Patterson B K et al Repertoire of chemokine receptor expression in the female genital tract implications for human immunodeficiency virus transmission Am J Pathol 153 1998 481 490 Pongers Willemse M J Verhagen O J Tibbe G J Wijkhuijs A J de Haas V Roovers E van der Schoot C E and van Dongen J J Real time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes Leukemia 12 12 2006 2014 1998 Pugazhenthi S Nesterova A Sable C Heidenreich K A Boxer L M Heasley L E and Reusch J E Akt protein kinase B up regulates Bcl 2 expression through cAMP response element binding protein J Biol Chem 2000 Apr 14 275 15 10761 6 Raggi C C Bagnoni M L Tonini G P Maggi M Vona G Pinzani P Mazzocco K De Bernardi B Pazzagli M and Orlando C Real time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system Clin Chem 45 11 1918 1924 1999 Ringel M D Balducci Silano P L Anderson J S Spencer C A Silverman J Sparling Y H Francis G L Burman K D Wartofsky L Ladenson P W Levine M A and Tuttle R M Quantitative reverse transcription polymerase chain reaction of circulating thyroglobulin messenger ribonucleic acid for monitoring patients with thyroid carcinoma J Clin Endocrinol Metab 84 11 4037 404
43. 1 5 C per second Temperature uniformity 0 5 C measured 30 sec after the clock starts Long term stability and high reliability Support for multiple consumable formats see page 1 9 Several different modes of operation including 9600 mode and programmable temperature ramps see page 3 19 Reduced instrument downtime by allowing immediate replacement of the block see page 7 6 Easy access to the sample block for troubleshooting and maintenance see page 7 6 Optics System IMPORTANT Do not remove the cover to the 7900HT instrument Only a qualified Applied Biosystems service engineer may repair or adjust the internal components Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 5 Chapter 1 Product Overview Computer Functions System Requirements Hard Drive Partitions A Microsoft Windows compatible computer is required to operate the Applied Biosystems 7900HT Fast Real Time PCR System and the Zymark Twister Microplate Handler Through the SDS and Automation Controller Software the computer e Provides the interface for programming and operating the instrument e Coordinates the operation of the 7900HT instrument automation module and the bar code readers e Provides storage and management of the data produced by the 7900HT instrument e Provides the local area network LAN connection to the SDS Enterprise Database if available The system requirements for the compu
44. 12 4042 4044 1999 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Bibliography 3 Smith G J et al Fast and accurate method for quantitating E coli host cell DNA contamination in plasmid DNA preparations BioTechniques 26 1999 518 526 Wang S C Klein R D Wahl W L Alarcon W H Garg R J Remick D G and Su G L Tissue coexpression of LBP and CD14 mRNA in a mouse model of sepsis J Surg Res 76 1 67 73 1998 Wang T and Brown M J mRNA Quantification by Real Time TaqMan Polymerase Chain Reaction Validation and Comparison with RNase Protection In Process Citation Anal Biochem 269 1 198 201 1999 Wang X X Li J A Erhardt F C Barone and G Z Feuerstein 2000 Detection of tumor necrosis factor alpha mRNA induction in ischemic brain tolerance by means of real time polymerase chain J Cereb Blood Flow Metab 2000 Jan 20 1 15 20 20 15 20 Wang X X Li R W Currie R N Willette F C Barone and G Z Feuerstein 2000 Application of real time polymerase chain reaction to quantitate induced expression of interleukin 1 beta mRNA in ischemic brain tolerance J Neurosci Res 2000 Jan 15 59 2 238 46 Ying H T Z Zaks R F Wang K R Irvine U S Kammula F M Marincola W W Leitner and N P Restifo Cancer therapy using a self replicating RNA vaccine Nat Med 5 823 827 1999 Relative Araki R and et al Nonsense mutation at Tyr 406 in the DNA depe
45. 17 18 19 20 Defining the 1 Positions of the 2 Remaining Stacks Aligning the Plate Handler Click the Yertical Home While holding the plate click Open Gripper and remove the plate In the Vertical Adjustment field enter 3200 and press Enter The Plate Handler lowers the arm to a position near the base of the output stack Carefully lower the Plate Handler arm until it is approximately 1 2 mm from the bottom of the stack Click f Vertical arser click s and record the number in the Vertical Adjustment field The software records the vertical position for position 0 the output stack Click t Vertical Heme The Plate Handler raises the Plate Handler arm to its highest position In the Vertical Adjustment field enter the Vertical Offset value determined in step 17 and press Enter The Plate Handler lowers the Plate Handler arm to a Vertical Offset position If necessary readjust the Vertical Offset value and repeat steps 18 through 19 until satisfied with the setting Place an empty plate into input stack 2 Zymark position 5 Inthe Zymark Twister Software click position 5 The Plate Handler arm moves over the input stack Using the Vertical Positioning commands lower the Plate Handler arm until it is approximately 1 cm above the stack and center it using the Rotary Adjustment arrows Carefully lower the Plate Handler arm into the stack Center the gripper as it moves down
46. 2 Inthe Save Document as Template dialog box click tes 3 4 Inthe Template Name field of the Save Template to Database dialog box enter If the software displays one or more warnings read the note and click __ _ a name for the plate document template up to 128 characters and click Select File gt Close If the software prompts you to save the plate document click m The SDS software closes the plate document template Create a plate document from the plate document template as explained on page 3 24 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 23 Chapter 3 Preparing a Run Step 7 Creating a Plate Document from the Template User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to create a plate document from a plate document template Options for The SDS software offers two options for creating plate documents from a plate Creating Plate document template individually or in batches Documents from the Template Option Description See Page Create an individual plate The procedure below explains how to Follow the document from the plate create a single plate document from a procedure document template plate document template for running a below plate By repeating the procedure you can create as many plate documents as needed Create multiple plate documents As a faster al
47. 22 showing 2 21 Port Number A 22 Post readout from the Plate Read tab 4 27 Pre readout from the Plate Read tab 4 27 precision causes of low precision 8 5 to 8 8 preparing master mixes B 3 plate forarun 4 6 primer and probe concentrations optimizing B 3 programming methods absolute quantification 3 19 methods allelic discrimination 3 18 methods dissociation curve analysis 3 21 methods relative quantification 3 19 temperature ramp 3 21 Protocol preventing contamination 4 6 pure dye plate constructing for custom dyes 7 27 preparing foruse 7 24 pure dye run about 7 20 creating a plate document 7 21 extracting data 7 25 performing 7 24 troubleshooting 8 11 when to perform 7 20 Q Quality control TaqMan Low Density Array 4 12 quantifying probes and primers B 3 standards for absolute quantification B 6 quantitative RT PCR about 6 3 absolute See absolute quantification relative See relative quantification types of 6 3 quantities applying to a plate document 3 15 R R2 readout from the Standard Curve Plot 6 13 reagents custom oligonucleotides C 6 non Applied Biosystems PCR reagents 8 8 TaqMan Pre Developed Assays and Reagents C 6 TaqMan RNase P Instrument Verification Plates C 5 re connecting the SDS software 4 30 relative quantification about 6 3 6 16 to 6 20 analyzing data 6 22 to 6 35 assay development guidelines B 6 procedure checklist 6 22 the Comparative Cy Method 6 3 the Standar
48. 3 Dissociation Curve Analysis ccc cece cece cc cccces 6 37 O n be ko be 8465550 65636650 6508S robs bead hae ensdseedeseees 6 38 Dorie YO GO kg og eye eR RT OE ENE RE EEE AS ORE ERS 6 39 Analysis Checklist oo vd cee see ets e 656049 49 0 40554 OEHO Se eS re oeears 6 40 APU mE Ee boone ed on bes 4 EEEE aa es 6 41 Analyzme Te Rin A v ksod ew sda none a doa een Phew byob bes use bon ns 6 41 Determining T Values for the Analyzed Run annanaannanna anaana 6 42 Alter eh os 5 ok ee ee ee 6 Gs Oe OG Ses 6 44 Section 6 4 Procedure Reference ccc cece cece re ere cceee 6 45 Setting the Baseline and Threshold Values 0 0 0 0 cc cece eee 6 46 Eliminating Outlying Amplification 445644 o4 440 95404446 e ein ee ee eeens 6 49 After the Analysis 0 0 0 0 00 ee eee teen ens 6 52 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 1 Chapter 6 Analyzing Real Time Data Notes for Database Users Database Alerts and Notifications Reason s For Change Dialog Box Electronic Signature Verification Dialog Box If you are using an SDS Enterprise Database to store SDS plate documents sessions studies and data you may be required to perform additional tasks when analyzing data as described in this chapter This section describes the types of actions you may need to perform depending on how your administrator has configured the database For more information o
49. 5s 4a E eho 6G 4 405654 ess HHA eo Se eh bKS 49 e EH 3 3 VWOLKIIOW VOIO 4 4544 64446555 OS SADA RY RAS Oh de E40 Od ESS 3 4 Quick Review Powering On the 7900HT Instrument 4 3 7 Step l Creating a Plate Dac ss eo 5 60 664 0616 Oe ee es 3 8 Step 2 Applying Detectors and Markers 0 cc cece ee eee 3 9 Step 3 Configuring the Plate Document with Tasks 3 14 Step 4 Setting the Passive Reference and Omitting Wells 3 16 Step 5 Programming the Plate Document Method 3 17 Step 6 Saving the Plate Document as a Template 0 3 22 Step 7 Creating a Plate Document from the Template 3 24 Step 8 Applying Sample and Plate Information 05 3 25 Step 9 Running the Plate on the 7900HT Instrument 3 26 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 1 Chapter 3 Preparing a Run Notes for Database Users Database Alerts and Notifications 3 2 Reason s For Change Dialog Box Electronic Signature Verification Dialog Box If you are using an SDS Enterprise Database to store SDS plate documents sessions studies and data you may be required to perform additional tasks when creating and modifying plate documents as described in this chapter This section describes the types of actions you may need to perform
50. 8 8 Optical 96 Well Fast Thermal Cycling Plates C 4 Optical Adhesive Covers C 3 Optical Cap Strips C 3 writing on reaction plates 8 7 Contamination genomic DNA 4 11 preventing 4 6 contamination decontaminating the sample block 7 14 Index 2 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide fluorescent common sources 8 7 isolating on the sample block module 8 9 contextual menus using 2 28 Control assay 4 11 copying detectors to a plate document 3 11 markers to a plate document 3 13 Cover readout from the Real Time tab 4 27 creating background plate documents 7 17 custom pure dye plate documents 7 28 detectors 3 9 markers 3 12 plate documents 2 13 3 8 plate documents from a template 3 24 4 36 pure dye plate documents 7 22 Cr See threshold cycle custom pure dyes adding to the pure dye set 7 27 to 7 29 creating a custom pure dye plate 7 27 cycle set adding toamethod 3 19 D data type definitions exportable A 17 database saving plate documents 4 24 saving results 5 19 6 53 saving templates 3 23 database connection A 22 decontaminating the sample block module 7 14 to 7 15 defragmenting the hard drive 7 54 deleting steps from a method 3 19 detector tasks about 3 14 applying 3 14 importing into a plate document A 2 detectors about 3 9 applying to a plate document 3 11 copying toa plate document 3 11 creating 3 9 importing into a plate document A 2
51. 99 9 C Time field 0 01 to 98 59 min 1 Click the step to the left of the location you want to place the new stage 2 Click Add cycle or Add Hold The software inserts the stage into the thermal profile Note To add a step to a stage select the step to the left of the location you want to place the step and click Add Step Thermal Profile Auto Increment Ramp Rate Data Collection Stage 1 Stage 2 Stage 3 Selected step Repeats lao 96 0 New step appears here remove a step 1 Click the step you want to remove The software highlights the selected step 2 Click __Delete Step to remove the step from the profile Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 19 Chapter 3 Preparing a Run 4 Configure the data collection options for the method a Select the Data Collection tab b Click below each plateau or ramp inside the cycle stage of the thermal profile to place a data collection icon at each step Thermal Profile Auto Increment Ramp Rate Data Collection Stage 1 Stage 2 Stage 3 Cycle 0 of 40 95 0 95 0 Data collection icons IMPORTANT Configure the method of your plate document to collect data only in the necessary stages of the PCR The file size for a real time run depends on the length of the run and the configuration and placement of the data collection icons
52. ARERR RRR RRR ttt ttt tt EE A l PH At B ot D oo ot oo ea a D O A O E Figure 6 5 Components of the Standard Curve Plot Starts the Sequence Detection Systems Software Online Help e Detector menu Toggles the data displayed within the plot based on detector name e Legend box Displays a symbol key for the datapoints appearing in the plot e Hide Unknowns button Toggles the presence of data from unknown samples in the plot e Standard Curve box Contains the following statistical data describing the standard curve Slope The slope of the standard curve The slope of the standard curve is useful for assessing the efficiency of the assay At 100 efficiency a reaction should achieve a slope of 3 33 since every 10 fold difference in quantity translates to a difference of 3 33 Cys Y Inter The Y axis intercept of the standard curve R2 The R square value for the standard curve that describes the correlation between threshold cycles Cy and the log of the quantity value for the samples that comprise the standard curve plot The calculation yields a value between and 0 where values closer to 1 indicate better correlation between Cz and the log of the quantity value Note The software calculates the R square value by taking the square of the Pearson Coefficient of Correlation also known as the r value calculated for the data points that comprise the plot The software calculates the R
53. C If you start a run before the heated cover reaches 105 C the instrument will pause until it reaches the optimal temperature before commencing the run 1 Power on the monitor and computer 2 Power on the Zymark Twister Microplate Handler by pressing the power switch located on the back panel of the Plate Handler see below ASIII PHYSICAL HAZARD Keep clear of the arm when activating the Plate Handler Once activated the arm automatically moves to its home position HI POT A cam O Rear Panel of the Twister F lt Power switch If operating normally the Plate Handler moves the arm to the home position over the output stack 3 Power on the 7900HT instrument by pressing the power button located below the status lights on the front of the instrument see Figure 2 1 If operating normally the 7900HT instrument will do the following on startup e Emit a high pitched tone signaling that the instrument initialized successfully Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Powering On the 7900HT Instrument e Cycle the status lights Red Orange Green indicating that the 7900HT instrument is active see Reading the Instrument Status Lights on page 2 5 for more information IMPORTANT Do not power on the 7900HT instrument if you have removed the lower side panel of the instrument Doing so
54. C for quantitative PCR and 65 67 C for allelic discrimination using the Primer Express software Select the strand that gives the probe with more Cs than Gs For allelic discrimination see page B 5 Adjust probe length so that both probes have the same T Position the polymorphism site approximately in the center of each probe For absolute or relative quantification see page B 6 For multiplex PCR applications involving multiple probes design the probes with different fluorescent reporter dyes see Table B 1 B 2 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Assay Development Guidelines Table B 1 Reporter Dyes for Multiplex PCR Applications First Probe Second Probe Reporter Dye FAM VIC The use of the FAM and VIC reporter dyes for multiplex applications provides the greatest degree of spectral separation Design Primers for the Assay Adhere to the following guidelines when designing primers for 5 nuclease assays e Keep the G C content in the range of 30 80 e Avoid runs of an identical nucleotide especially guanine where runs of four or more bases should be avoided e Keep the T in the range of 58 60 C using the Primer Express software e Limit the G and or C bases on the 3 end The five nucleotides at the 3 end should have no more than two G and or C bases e Place the forward and reverse primers as close a
55. CO oy on dane ewes 645 95 Oo Fae bse eae ke 5004 ee 84 EES 6 39 Pa eo 5 5h hb a5 2 oe oe SS Ses oe oh oe ES 6 40 Opening the Run Dats 6 i400 b dad naana bane eee ee edd ew ee bee HOE RES 6 4 Analyzing the R n DA 4 6 iin tee 6404049 aE a ead bee ee eden ee ees RANEY 6 41 Determining T Values for the Analyzed Run 0 200002 ce ee 6 42 After tbe Analysis y 5 5 44 55 64 0 6044 64014 6656445 0b 04 A645 944449 4 ROE 6 44 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 37 Chapter 6 Analyzing Real Time Data Overview About Dissociation Curve Analysis Employing the SYBR Green 1 Dye Mathematical Transformations The Applied Biosystems 7900HT Fast Real Time PCR System supports dissociation curve analysis of nucleic acids using SYBR Green 1 double stranded DNA binding dye chemistry The objective of dissociation curve analysis is to accurately determine the melting temperature T of a single target nucleic acid sequence within an unknown PCR sample Typical uses of dissociation curves include detection of non specific products and primer concentration optimization Dissociation curve analysis on the 7900HT instrument is made possible through the use of the fluorogenic SYBR Green double stranded DNA binding dye chemistry see page D 3 Dissociation curves are commonly performed following the PCR stage of a SYBR Green dye experiment to screen for non specific products To generat
56. Click or select Analysis menu gt Analysis Settings 2 In the Detector drop down list select a detector 3 Inthe Analysis Settings dialog box configure the method automatic or manual that the software will use to determine the baseline and threshold values for the selected detectors Analysis Settings Absolute Quantification m Ct Analysis p Detector ote ei a Automatic Ck amp Manual Ck Threshold 0 20 Automatic Baseline f Manual Baseline start 2 Stop 3 a Apply OK Cancel To determine the baseline and threshold values Automatically Select the Automatic Cy option button IMPORTANT If you choose to use the AutoC algorithm verify that the baseline and threshold were called correctly for each autocalled detector following the analysis use the procedure on page 6 46 Note See Automatic Threshold and Baseline Determination on page 6 18 for more information on the AutoC algorithm Manually Do the following a Select the Manual Ct option button b In the Threshold field enter a threshold value to apply to the selected detector s or leave the field empty and set the threshold value manually as explained on page 6 46 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 11 Chapter6 Analyzing Real Time Data c Do one of the following Select the Automatic Baseline option bu
57. Click ok to continue Description of the action that requires a signature Enter your user name Enter your password Figure 6 2 Electronic Signature Verification Dialog Box Options 6 2 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Real Time Runs on the 7900HT Instrument Real Time Runs on the 7900HT Instrument Real Time Runs Real time is the term used to describe the category of sequence detection runs in which the Applied Biosystems 7900HT Fast Real Time PCR System is used to measure the fluorescence of a biological sample during thermal cycling In contrast to end point runs real time experiments can be used to achieve both qualitative and quantitative measurements Real time analysis can be used in combination with either TaqMan or SYBR Green 1 double stranded DNA binding dye reagents for a variety of purposes including quantitative PCR and dissociation curve analysis Quantitative Quantitative RT PCR is a method used to measure small quantities of ribonucleic RT PCR acid sequences isolated from biological samples Typical biological samples include cells tissues and fluids During the RT step reverse transcription of target RNA produces corresponding complementary DNA cDNA sequences During the subsequent PCR the initial concentration of target cDNA is quantified by amplifying it to a detectable level The two types of quantitative RT PCR supported by the 7900HT ins
58. Database SDS Document Extractor Archiver Archive Files arc Figure 1 17 Functional Summary of the Database Management Utilities The SDS Enterprise Database Software Suite includes four command line utilities for managing plate documents plate document templates and run data produced by the 7900HT instrument SDS Document Creator Creates plate documents in the database using the contents of a plate document template and an assay plate setup file SDS Document Loader Populates the database with the contents of existing plate documents SDS Document Extractor Creates plate documents from the data contained in the database SDS Template Loader Populates the database with the contents of existing plate document templates For detailed information on these utilities see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide PN 4351669 The SDS Archiver Software installs with the version of the SDS software that includes the database client software The software allows users with Administrator privileges to download session run data from the database create archive files from them and restore archived data to the database See the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide PN 4351669 for more information Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Da
59. Exporting Data from a Plate Document on page 2 14 into the empty plate document created in Exercise 1 Creating a Plate Document on page 2 13 1 Ifthe plate document from Exercise 1 is still open go to step 2 Otherwise create a new plate document to receive the setup table data a Click or select File gt New b Configure the New Document dialog box with the same settings as the plate document template Assay drop down list Select Absolute Quantification Container drop down list Select 96 Wells Clear Plate Template drop down list Select Blank Template Barcode field Leave blank c Click x The software displays a new plate document with appropriate attributes 2 Click or select File gt Import 3 In the Look In field of the Import dialog box navigate to Applied Biosystems SDS Documents 4 Select the Practice txt file created in Exercise 5 GIWeEeG Ee m 2 File name Practice txt Import sail Network Files of type Tab delimited Text txt Cancel 5 Click import The software imports the setup information of the Practice txt file into the plate erid and table of the empty plate document Note For more information on importing and exporting setup table data using the SDS software see Appendix A Software Reference 6 Save the plate document as explained on page 2 17 2 16 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Ent
60. General Instrument Safety ecc oh ode heehee heed eed be bees oes xvii Chemical GAIN 2 u4 2 4 006 adad aei aa a a sade s i E eee e xviii OA Wae Day ok oe eh bes ee eee EERTE AEDE EEE EE XIX aes chosen wide ennvune E R E sb adandat Hehe aage ened XX Physical ee ec os hon ee heme ees ee oe oP RO ERR on eed eo XXi Biological Hazard Sally eunea asarni an eE na E E A ES xx Larr A peas be eh ee ee eee bee eee ee ee eee ee es XX1 BarCode ncanner Laser Salei 2 sk aanceat Ve nana eens Gee een a chien A xxii Workstafion Salely oer ee deg eees ee EEES EEEE NEN E EEEE EERE xxiii Safety and Electromagnetic Compatibility EMC Standards xxiii Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide xiii Safety and EMC Compliance Information Safety Conventions Used in This Document Safety Alert Four safety alert words appear in Applied Biosystems user documentation at points Words _ inthe document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical N 07l Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices ANITY
61. Plate Handler raises the arm to its highest position If the plate contacts the sides of the stack re adjust the rotary position of the Plate Handler arm until the plate moves freely inside the stack Note Contact between the plate and the stack or all stacks may be unavoidable However try to minimize the contact as much as possible Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 45 Chapter 7 Maintaining the Instrument 9 Click Rotary onset and click es The software re records the rotary position for the input stack 1 Zymark position 4 While holding the plate click pen Gripper and remove the plate Defining the The Automation Controller Software requires a bottom position value for all stacks Bottom of the This value is used to prevent the Plate Handler arm from colliding or grinding as it Stack moves to the bottom of each stack l 2i 3 10 11 12 Remove all plates from the instrument and the Plate Handler arm Place an empty plate into the output stack Zymark position 0 In the Zymark Twister Software click position 0 The Plate Handler arm moves over the output stack Using the Vertical Positioning commands lower the Plate Handler arm until it is just above the stack Check the rotary position of the Plate Handler arm to confirm that the gripper e is centered over the stack e will not contact the sides of the stack when lower
62. R for Cy determination in real time assays The level is set to be above the baseline and sufficiently low to be within the exponential growth region of the amplification region of an amplification curve The threshold is the line whose intersection with the Amplification plot defines the C For a given well the threshold cycle C represents the PCR cycle at which the SDS software first detects a noticeable increase in reporter fluorescence above a baseline signal 1 Chemical The temperature at which 50 of the DNA amplicons are in a double stranded configuration 2 Mathematical The maximum value for the first derivative curve of normalized reporter fluorescence R Glossary 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Index Symbols 6 31 T 6 33 Numerics 7900HT instrument See instrument 9600 emulation mode 3 19 A ABI PRISM 7700 Sequence Detection System emulating 3 19 ABI PRISM Sealer see Sealer absolute quantification about 6 6 to 6 7 analyzing data 6 9 to 6 13 assay development guidelines B 6 procedure checklist 6 9 selecting and preparing standards B 6 troubleshooting 8 12 adding bar code to a plate document 2 27 3 25 custom dyes to the pure dye set 7 27 detector tasks to a plate document 3 14 detectors to a plate document 3 11 markers to a plate document 3 13 plate documents to the plate queue 4 35 4 40 sample names to a plate document 3 25 a
63. Starting template copy number e Efficiency of DNA amplification the PCR system How the SDS Software Determines C s To determine the C for an Amplification plot the SDS software uses data collected data from a predefined range of PCR cycles called the baseline the default baseline occurs between cycles 3 and 15 First the software calculates a mathematical trend based on the baseline cycles R values to generate a baseline subtracted Amplification plot of AR versus cycle number Next an algorithm searches for the point on the Amplification plot at which the AR value crosses the threshold setting the default threshold setting is 0 2 The fractional cycle at which the intersection occurs is defined as the threshold cycle Cy for the plot Note It may be necessary to adjust the baseline and threshold settings to obtain accurate and precise data For further information on resetting the baseline and threshold settings see Setting the Baseline and Threshold Values on page 6 46 D 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Real Time Data Analysis Significance of Beginning with the equation describing the exponential amplification of the PCR Threshold Cycles n m X X 1 Ey where Xh number of target molecules at cycle n so that n gt m Xm number of target molecules at cycle m so that m n Ey efficiency of target amplification between 0 1 n m
64. Time PCR System and SDS Enterprise Database User Guide Replacing the Sample Block Materials e Replacement Sample Block if replacing the sample block Required e 5 32 inch hex key necessary only for certain instruments e 5 16 inch hex key some instruments may require a crescent wrench Handling the The interchangeable sample blocks are delicate pieces of equipment containing Sample Block several fragile components that can break if handled improperly Figure 7 3 shows the correct locations for handling the interchangeable sample block module Circuitry and connections to the instrument Do Not Touch Heat sinks Hold sample block Bottom of module module from the sides Figure 7 3 Locations for Handling Sample Block Modules Removing the 1 Start the Automation Controller Software then Sample Block a Select the Thermal Status tab then confirm the function of the current module The module is operating normally if the software is receiving a temperature reading b Click openjctose to rotate the instrument tray to the OUT position 2 Select File gt Exit to close the Automation Controller Software 3 Power off and unplug the 7900HT instrument Nm PHYSICAL HAZARD The instrument must be unplugged and powered of
65. To use this table look for the category and the symptom you are experiencing The page number in the right hand column corresponds to a description of the possible cause s and recommended action s for that particular problem Table 8 1 Troubleshooting Table Category Symptom Page Chemistry and Run Problems Chemistry Low Precision 8 5 Irreproducibility Software Installation SDS Software Unable to finish installation 8 5 Install program appears to b e frozen Installation Interrupted Run Problems Background Runs Software will not extract background data Background is too high greater than 2500 Pure Dye Runs Software will not extract pure dye data 8 11 Raw data from pure dye run appears strange Signals plateau saturation Signal is too low lt 10 000 FSU More than two outliers per dye in a single row Real Time Runs Quantitative PCR and Dissociation Curves 8 12 End Point Runs Allelic Discrimination 8 13 8 2 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Troubleshooting Table Table 8 1 Troubleshooting Table Category Symptom Page Instrument and Automation Accessory Problems Software and 7900HT SDS software will not start 8 14 Instrument Software crashes freezes the computer or displays an error message Communication error Thermal cycler errors
66. Users 2 0 cc eee 2 11 Lesson 1 Using Plate Documents 0 0 ee ees 2 12 Lesson 2 Viewing and Resizing PaneS 0000 eee eee 2 21 lesson 3 Using the Plate Grid c e aiaiee dee le eae ee hehe es 2 23 Lesson 4 Using the Hand Held Bar Code Reader 002 eee aes 2 27 Lesson 5 Using Contextual MenuS 0 0 eee ee eee 2 28 Lesson 6 Using Keyboard Shortcuts 2 0 0 eee 2 28 Using SDS Plate Documents 2 00 eee tees 2 29 Preparing a Run Notes f r Database Users 201i oes cede onde eee ee She ee eed dene ete 3 2 Before YOU BEGIN rsca a ete Mids kobe BOY id 2 herd we baie E eee Ge 3 3 Workflow OvervieW Aione aa a e ee et ee eee ete 3 4 Quick Review Powering On the 7900HT Instrument 2000 eee 3 7 Step 1 Creating a Plate Document 0 00 tees 3 8 Step 2 Applying Detectors and Markers 000 cee eee eee ees 3 9 Step 3 Configuring the Plate Document with Tasks 0002 eee eeee 3 14 Step 4 Setting the Passive Reference and Omitting Wells 3 16 Step 5 Programming the Plate Document Method 0000 eee eee 3 17 Step 6 Saving the Plate Document as a Template 0 000 c eee eee 3 22 Step 7 Creating a Plate Document from the Template 00055 3 24 Step 8 Applying Sample and Plate Information 0 00 eee eee eee 3 25 Step 9 Running the Plate
67. a noticeable rise in fluorescent signal during the entire PCR the fluorescent signal produced by the well never crosses the threshold level defined for the associated detector AC Displays the normalized threshold cycle for the sample AvgAC Displays the averaged normalized threshold cycle for the sample replicate group AC SD Displays the number of standard deviations from the average AC of the replicate group that the sample s individual AC value lies AAC Displays the calculated AAC value for the replicate group associated with the test sample RQ Displays the calculated relative level of gene expression for the replicate group associated with the test sample RQ min Displays the minimum calculated relative level of gene expression in the test samples RQ max Displays the maximum calculated relative level of gene expression in the test samples Status When autocalling is enabled and a sample 1s reject by the algorithm the status field indicates how the sample failed the analysis Rejected When autocalling is enabled indicates if the sample data was rejected 6 34 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide After the Analysis After the Analysis User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to save analyzed data from a relative quantification study Post A
68. analyzing the data for your study you must specify the analysis settings for each detector that you want to analyze The settings are detector specific and must be set for each detector individually The following sections briefly discuss the analysis settings provided by the software in the Analysis Settings dialog box e About the Analysis Settings see below e Specifying the Analysis Settings see page 6 29 Note You can analyze the run without configuring the analysis settings However the software analyzes the data using the baseline of cycles 3 to 15 and a default threshold value of 0 2 About the Analysis Settings C Analysis for Selected Detector Settings The settings of this group box control the method that the SDS software uses to calculate the Cys of the sample replicate groups associated with the selected detector in the Detector drop down list As shown in Figure 6 9 the Ct Analysis settings provide both automatic and manual methods for determining the baseline and threshold values Automatic Ct option button Manual Ct omen Manual Ck i aE Threshold 0 20 option button Automatic Baseline f Manual Baseline star 3 4 stop 15 4 Figure 6 9 C Analysis for Selected Detectors Settings e Automatic Ct Instructs the software to calculate the optimal baseline range and threshold values for the selected detectors using the AutoC algorithm see page 6 18 IMPORTANT If you choose to us
69. as a single data point on the plot Figure 5 6 illustrates the components of the Allelic Discrimination plot E HTC w Undetermined Marker Call drop down list drop down list Toolbar Setup Instrument Results Marker CYP 2 Call k X al a El Scatterplot x ir z Legend a Allele X D Allele Y Allele 277 2 2 z 1 0 o 0 1 0 20 3 0 40 Allele X CYP 2C19 2 2 Figure 5 6 Components of the Allelic Discrimination Plot e Marker drop down list Determines the marker data that the software displays within the plot e Call drop down list When a datapoint is selected this menu allows you to assign an allele call to the datapoint within the scatterplot Toolbar Contains the following tools for manipulating the plot Icon Description h Selects individual data points by clicking or groups of datapoints by clicking and dragging a box across a group of data points Selects groups of datapoints by encircling them with the tool Repositions the view within the plot by clicking and dragging the screen a Zooms the plot by clicking the mouse button within the plot or by clicking and dragging a section of the plot to view a Zooms out on the plot by clicking the mouse button within the plot e Scatterplot A scatterplot of data points from the run Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database Use
70. both the SDS software and the computer operating system As the hard drive becomes fragmented programs take greater time to access files because they must perform multiple seek operations to access the fragments 7 54 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Upgrading the Operating System Software Maintaining the SDS software Several commercially available software utilities are available for repairing fragmented file systems The software utility defragments broken files by combining their component pieces at a single location on the hard drive thereby optimizing system performance Do not upgrade the operating system of the computer connected to the 7900HT instrument unless instructed to do otherwise by an Applied Biosystems representative New versions of the Microsoft Windows operating system can be incompatible with the SDS software and render it and the instrument inoperable Applied Biosystems service engineers maintain the operating system software as part of planned maintenance visits During the visit an engineer will update the computer operating system as upgrades become available and are validated by Applied Biosystems Maintaining the SDS software User Access Requirement Upgrading the SDS Software Reinstalling the Software IMPORTANT You must have administrator privileges on the computer to install and or upgrade the SDS software Applied Biosystems con
71. change in fluorescence as a function of temperature see below R n i dT The software plots the negative of the resulting derivative data on graph of R versus temperature 7 that visualizes the change in fluorescence at each temperature interval The T for the target nucleic acid can be determined from the graph by identifying the maximum for the rate of change displayed as a peak for the appropriate amplification curve 6 38 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Before You Begin Example Results Figure 6 14 illustrates a typical dissociation curve from an experiment run to detect non specific amplification in cDNA samples The figure displays the dual amplification peaks typical of primer dimer formation The amplification from the specific product is displayed with a T of 80 5 C while the primer dimer product has a characteristically lower T of 74 9 C 6 0 Main Product N T 80 5 C 5 0 4 0 3 0 2 0 1 0 0 0 XA 60 65 70 75 80 85 90 95 Temperature C Figure 6 14 Example of Results from a Dissociation Curve Analysis Before You Begin Using the SDS For specific instructions on any procedure described within this section refer to the Software Sequence Detection Systems Software Online Help To get help at any time during the Online Help procedure click 17 located within the dialog box or window in which you are working E
72. click Stop in the Automation Controller Software dialog box at any time If you clicked top E Then the instrument while the Plate Handler is aborts the run and moves the Plate Handler to the home handling a plate position after a plate has been loaded aborts the current run ejects the plate and moves the into the instrument but Plate Handler to the home position before the run has started after the 7900HT instrument aborts the current run ejects the plate and moves the has started a run Plate Handler to the home position IMPORTANT Stopping a run during thermal cycling can affect the chemistry of the reactions inside the plate Before stopping a run carefully read the guidelines on page 4 28 to determine the best course of action To eject a plate following a halted run or to open close the instrument tray click openjciese from the Automation Controller Software window You can analyze the run data immediately following the completion of the run See the appropriate page for your application Section 5 1 Allelic Discrimination 0 0 0 0 0 0c cece ee eee 5 5 Section 6 1 Absolute Quantification 644441 4h6400sh04sereuseed caw se edsas 6 5 Section 6 2 Relative Quantification 4444 i065 450405448 400545 4446 6404085 6 15 Section 6 3 Dissociation Curve Analysis 0 0 0 0 00 ccc ccc eee 6 37 4 44 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User
73. code reader 7 49 aligning the Plate Handler 7 41 performing background run 7 16 performing pure dye spectral run 7 20 calibrator sample 6 20 Centrifuge system bucket requirements 4 16 Bucket Type setting 4 13 components 4 12 installation and operation 4 13 placing bucket in centrifuge 4 17 requirements 4 12 TaqMan Low Density Array placing in bucket 4 16 Centrifuging TaqMan Low Density Array 4 16 changing gripper finger pads 7 52 pane view and plot sizes 2 21 sample block module 7 6 checklists absolute quantification 6 9 allelic discrimination 5 10 dissociation curve 6 22 6 40 relative quantification 6 22 chemistry 5 Nuclease TaqMan D 2 non optimized 8 6 SYBR Green D 3 troubleshooting 8 5 to 8 8 cleaning gripper finger pads 7 52 sample block wells 7 15 closing instrument tray Automation Controller Software 4 44 instrument tray SDS software 4 29 plate documents 2 18 Zymark Twister Software 7 48 comments adding to a detector 3 9 adding to a plate document 3 25 Comparative Cy Method about 6 3 D 8 formula derivation D 8 Components centrifuge system 4 12 TaqMan Low Density Array 4 10 computer about 1 6 hard drive partitions 1 6 maintaining 7 54 minimum system requirements 1 6 troubleshooting 8 14 turning ON 2 4 configuring the 7900HT instrument for 9600 emulation 3 19 consumables 384 Well Optical Reaction Plates C 3 96 Well Optical Reaction Plates C 4 improper or damaged plastics
74. database before a user belonging to the Operator User Group can run it Options The 7900HT instrument can run prepared plates individually or in groups using the for Running Zymark Twister Microplate Handler SDS Plates IMPORTANT If you are not using a Zymark Twister Microplate Handler you must run plates individually Choose one of the following options to run the plate To run the plate Description See Page individually 1 Prepare the consumable optical plate or 4 5 TaqMan Low Density Array 2 Run the plate array individually using the SDS 4 23 software as part of a group 1 Prepare the consumables optical plates or 4 5 Automated Operation TaqMan Low Density Arrays 2 Run the plate array with others as part of a batch 4 31 from the Automation Controller Software using the Zymark Twister Microplate Handler IMPORTANT You must have a Zymark Twister Microplate Handler to run plates using this option 3 26 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Operating the Instrument In This Chapter Notes for Database Users 000 cence e nen n ees 4 2 Pe OS op 4s nooo pao h5 548 Fo wees se hha ee owe 49 ES 4 3 Section 4 1 Consumable Preparation ccc cece cece ecccccees 4 5 Prev CAs 9 oe ee wed os 59 ho 95 0845 be 4 Pees ees 4 6 Compatible Consumables 5 oie 54 6 4 0 h 4304 95155609 0509544405 094 4000484 4 6 Preparing Optical Plates fo
75. depending on how your administrator has configured the database For more information on any of the features described below see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide The Reason s for Change dialog box appears only if the SDS Enterprise Database is configured for auditing The auditing system monitors the creation modification and deletion of the SDS data contained by the database When the Reasons for Change dialog box appears do one of the following e Select a description for the change from the drop down list then click ox e Enter a description of the reason for the change in the field then click Reasons For Changa Reasons for change drop down list Resson For Change Attribute AGPlata Cd vee New Wale Default descriptions Calculation Error Meed to Change Threshold Example of a custom description for a change Figure 3 1 Reasons for Change Dialog Box Options Enter ihe Feacofor Change Custom description of the reason for the change The Electronic Signature Verification dialog box appears only if the SDS Enterprise Database is configured to require electronic signatures The electronic signature system restricts and tracks user access of the SDS data contained by the database When the Electronic Signature Verification dialog box appears 1 In the User ID and Password fields enter your u
76. eee 2 27 Aligning the Fixed Position Bar Code Reader 0 0000 eee 7 49 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 7 Chapter 1 Product Overview Zymark Twister Microplate Handler Description Zymark Twister Microplate Handler Components Plate Positions Using the Zymark Twister Microplate Handler The Zymark Twister Microplate Handler coordinates plate handling for the 7900HT instrument permitting 24 hour unattended operation The arm provides a 310 degree rotational swing that permits access to the 7900HT instrument drawer up to five plate stacking areas and the fixed position bar code reader Zymark Twister Microplate Handler cross sectional view of the gripper Plate sensor Gripper Adjustment switch knob Fixed position Expansion Plate stack bar code reader stacks Figure 1 6 Components of the Zymark Twister Microplate Handler Bar code Zymark Twister Microplate Handler J we Al top view Figure 1 7 Plate Positions of the Zymark Twister Microplate Handler ASIII LASER HAZARD Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots Never look into the laser beam Remove jewelry and anything else that can reflect the beam into your eyes Protect others from exposure to the beam For information on the Zymark T
77. from instrument The disconnect option permits the simultaneous operation of both the the Instrument SDS software and the Automation Controller Software Because both programs control the Applied Biosystems 7900HT Fast Real Time PCR System one program must relinquish control of the 7900HT instrument before the other can be used to operate it To Then disconnect the 1 In the SDS software select the Instrument tab of the open software from plate document the instrument 2 Select the Real Time or Plate Read tab 3 Click Disconnect Note Once disconnected the software neither monitors nor controls the 7900HT instrument reconnect the software to reconnect to an open plate document once disconnected 1 Select File gt Close to close the plate document 2 Click or select File gt Open 3 In the Look In field navigate to and select the plate document of interest 4 Click pen Upon opening the plate document the software re establishes the 7900HT instrument connection to reconnect to an new plate document click E or select File gt New Upon creation of the plate document the software re establishes the connection with the 7900HT instrument 4 30 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 4 3 Automated Operation Section 4 3 Automated Operation In This Section Operating the Software with an SDS Enterp
78. information on user accounts 3 Select Tools gt Options 4 In the Options dialog box select the Database tab 5 Select or deselect the Enable Enterprise Options check box to activate or deactivate the database features 6 Click 7 When prompted enter your user name and password then click ox _ A 22 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Connecting SDS Software to the Database Reconfiguring the IMPORTANT You must have Administrative privileges to reconfigure the SDS Database Enterprise Database connection see the SDS Enterprise Database for the Applied Connection Biosystems 7900HT Fast Real Time PCR System Administrators Guide for more information on user accounts The connection to the SDS Enterprise Database can be reconfigured from the Options dialog box of the SDS software as explained below 1 Start the SDS software see page 2 7 for more information 2 In the Login dialog box enter a user name and password of a user account that belongs to the Administrator user group 3 Select Tools gt Options 4 In the Options dialog box select the Database tab 5 Configure the Current Selected Connection settings _Host Name field Enter the host name or IP address of the database Current Selected Connection Host Marne fi 6r 116 205 87 oo m Port Number field D a ee Enter the port number of the Service Name sas22 database connection
79. into the instrument tray with the A1 label in the rear left corner see below IMPORTANT Make sure to install the correct version of the plate adapter for the plate format you intend to use The plate adapters are labeled for the consumable format they support Well A1 Label 5 Replace and tighten the four screws in the order shown below IMPORTANT The order in which the screws are tightened is important to ensure proper alignment of the plate to the sample block inside the 7900HT instrument Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 13 Chapter 7 Maintaining the Instrument Decontaminating the Sample Block When to Perform Materials Required Cleaning a Low 14 Density Array Block The following procedure describes how to decontaminate the wells of a sample block module The procedure eliminates residual PCR related products including fluorescent labeled TaqMan probes Clean the sample block as often as needed IMPORTANT If you plan to use a cleaning or decontamination method other than the one in this manual check with Applied Biosystems first to ensure that the method will not damage the sample block module or the 7900HT instrument e 10 Sodium hypochlorite bleach solution e Isopropanol 100 percent pure e 5 32 in hex key e Cotton swabs Neer CHEMICAL HAZARD Sodium hypochlorite bleach is a liquid disinfectant that can be corrosiv
80. location Remove file entry from plate queue and add the file to the plate queue again Dialog box does not respond to mouse clicks or key strokes Java Runtime Error Click the close box of the dialog box to close it Run will not start No calibration file No background data in calibration file background run has not been performed No pure dye data in calibration file pure dye run has not been performed Calibration file does not contain pure dye data for a dye used on the plate document Calibration file was created on another instrument Perform background and pure dye runs See Performing a Background Run on page 7 16 and Performing a Pure Dye Run on page 7 20 Disk drive containing the plate document has less than 50 MB of free space Heated cover cannot reach running temperature because no plate loaded Instrument tray contains a plate Check the capacity of the destination drive If less than 50 MB of free space remains remove or archive existing data files see page 7 54 Open the instrument tray and check that the instrument contains a plate Output stack contains a plate or plates Remove all plates from the output stack of the Plate Handler before starting the queue Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Chapter 8 Troubleshooting Table 8 4 Troubleshooting Software and C
81. make up the set Are outliers present Then Yes Record the well numbers of the outlying wells then go to the next step No Go to the next step In the Detector drop down list select another detector and repeat steps 3 through 5 Repeat until each detector has been screened for outliers If analyzing a Relative Quantification Multiple Plate Document deselect the wells of the current plate then repeat steps 3 through 6 for every plate in the study Are outliers present on the plate Yes If outliers are present for any detector eliminate the outlying wells from the analysis by omitting them as explained in Eliminating Wells from the Analysis on page 6 50 No If no outliers are present do one of the following e If analyzing an Absolute Quantification Plate Document view the results of the run displayed within the Standard Curve plot and Results table Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 49 Chapter6 Analyzing Real Time Data e If analyzing a Relative Quantification Multiple Plate Document view the results of the run displayed within the Gene Expression plot and Results table Eliminating Wells The SDS software allows you to omit wells from the analysis If you have identified from the Analysis outliers or data you want to exclude from the analysis follow the procedure below to remove the corresponding wells from use
82. molecule cDNA is made from RNA through the action of the enzyme Reverse Transcriptase RNA is converted to cDNA because cDNA is more robust than fragile RNA and because DNA templates are required for many applications including PCR In real time mode fluorescent signal intensity is collected for the entire plate every 8 5 seconds Clipped data points are calculated by averaging the last three computed R normalized reporter signal data points collected during the extension phase of each cycle repetition Clipped data are presented as baseline uncompensated data R vs cycle data and baseline compensated data AR vs cycle data Some executable programs can be invoked in a DOS Command Prompt window and or from within a bat script file to run usually without a graphic user interface GUI Such programs may support user specification of parameters via a command line where those parameters would be entered after the name of the executable program Often these command line parameters are filenames directory paths or flag settings 1 A vessel used to contain reactions run on the 7900HT instrument a microplate or a TaqMan Low Density Array 2 A reagent kit chemical or disposable labware used as part of an assay The type of plate to be analyzed by the 7900HT instrument The types are ABI PRISM 96 Well Optical Reaction Plates ABI PRISM 384 Well Optical Reaction Plates Optical 96 Well Fast Thermal Cycling Plates and Taq
83. number of cycles elapsed between cycle m and cycle n Amplicons designed and optimized according to Applied Biosystems guidelines amplicon size lt 150 bp have amplification efficiencies that approach 100 percent Therefore E 1 so that X n n m X 1 1 n m X a 2 To define the significance in amplified product of one thermal cycle set n m 1 so that l X02 X l Therefore each cycle in the PCR reaction corresponds to a two fold increase in product Likewise a change in threshold cycle number of one must equate to a two fold difference in initial template concentration Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide D 7 Appendix D Theory of Operation Comparative C Method of Relative Quantification Arithmetic The Comparative Cy Method is similar to the Standard Curve Method However it Formula uses arithmetic formulas to achieve the same results for relative quantification The amount of target normalized to an endogenous reference and relative to a calibrator is given by 5 AAC Derivation of the The equation that describes the exponential amplification of PCR is Formula X X x 1 Ey where Xn number of target molecules at cycle n X Initial number of target molecules E efficiency of target amplification i number of cycles The threshold cycle C indicates the fractional cycle number at which the amount of amplified target reaches a fixed t
84. on the 7900HT Instrument 2000 ee 3 26 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Chapter 4 Operating the Instrument Notes for Database Users 0 0 0 c cc eee ees 4 2 Belrore YOU COI as mres ansaa anc Atala H Bian ti aed Moment Glade ane a E a Meath ak aera anes 4 3 Section 4 1 Consumable Preparation 000 cee eee 4 5 Preventing ComamiNatoN 26 c 0iebewkwe teeta eileen Sat ieke ehe eee 4 6 Compatible Consumables 0 0 00 cee ee eee eee es 4 6 Preparing Optical Plates for Use 0 0 ccc ees 4 8 Preparing TaqMan Low Density Arrays for Use 0 000 ccc eee 4 10 About the Low Density Arrays 0 0 ee eee ee 4 10 About the Centrifuge System 00 eee eens 4 12 ADOULTINE SEIO shai iea wets Kah ote ale 9 coe oe dk ee We a es 4 14 Loading the TaqMan Low Density ArrayS 000 cc ees 4 14 Centrifuging the TaqMan Low Density Arrays 0 000 eee 4 16 Sealing the TaqMan Low Density Arrays 0 0 0 cc ee 4 19 Section 4 2 Running an Individual Plate 0 0 es 4 23 Saving the Plate Document 0 00 eee ees 4 24 Running a Single Plate Using the SDS Software 0 0 eee ee 4 25 Aer INE RUN eer irae act Aerts seam haar ao E Gee odes aon eats Gee wate ean nal 4 29 Section 4 3 Automated Operation 0 00 es 4 31 Operating the Software with an SDS Enterprise Database
85. on the elements of the Real Time and Plate Read tabs click 7 and see the Sequence Detection Systems Software Online Help Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 27 Chapter 4 Operating the Instrument Stopping the IMPORTANT Read the following directions carefully before stopping a Run Using the run in progress SDS Software Stopping End Point Runs Allelic Discrimination In the Plate Read tab of the plate document click Stop Stopping Real Time Runs Absolute or Relative Quantification Has the Cover reading in the Real Time tab reached 104 C e No the heated cover temperature is below 104 C In the Real Time tab of the plate document click stop e Yes the heated cover temperature has reached 104 C The instrument is running the plate and has begun thermal cycling Determine a course of action from the options in the following table Reason for Stopping the Run Then Forgot to make a change to the plate do not stop the run document such as adding a detector detector task or sample name not including mistakes to the temperature profile Allow the instrument complete the run then edit the plate document before analyzing the data The software does not use detector or sample information until you analyze the plate document e Forgot to add a reagent to the plate 1 In the Real Time tab of the plate such as enzyme or master mix docu
86. optimal heat transfer Applied Biosystems recommends using compression pads when running the ABI PRISM 96 Well Optical Reaction Plates If you are going to run the plate e Individually using the SDS software then apply a standard compression pad e As part of a batch using the Automation Controller Software then apply an ABI PRISM Snap On Compression Pad IMPORTANT The ABI PRISM Snap On Compression Pad plate assembly may still be hot after the Zymark Twister Microplate Handler loads it into the Plate Handler s output stack Please wait at least 30 seconds before manually handling the Snap On Compression Pad plate assembly IMPORTANT Performance of the Optical 96 Well Fast Thermal Cycling Plates with the Zymark Twister Microplate Handler has not been verified Therefore when running multiple Optical 96 Well Fast Plates on the 7900HT instrument Applied Biosystems recommends that you load and unload the plates manually See Appendix C Kits Reagents and Consumables for a list of available consumables requirements and purchasing instructions Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 9 Chapter 1 Product Overview Instrument Connections Electrical Figure 1 8 and Table 1 2 illustrate the electrical connections of the Applied Connections Biosystems 7900HT Fast Real Time PCR System components
87. plate type setting A background plate was not run before the pure dye plate or card Run a background plate then run the pure dye plate or card again Raw data from pure dye run appears strange see below Raw Data Plot Calibration Data 3 00 E 4 2 50 E44 2 00 E 4 1 50 E 4 Amplitude Temperature C Pure dye plate or card was loaded backwards Raw Data Plot 17 ime 1 Verify the pure dye wavelengths are as expected 2 Rerun the pure dye plate or card Signals plateau saturation Signal is too low lt 10 000 FSU Intensity is set too high low Call Applied Biosystems Technical Support More than two outliers per dye in a single row e Evaporation e Contamination Rerun the pure dye plate or card If the problem persists discard the pure dye plate or card and runa new one Chapter 8 Troubleshooting Real Time Runs Quantitative PCR and Dissociation Curves Troubleshooting When faced with irregular data you can use the SDS software to diagnose some Analyzed Data chemistry and instrument related problems The following table contains a summary of checks for verifying the integrity of your run data and to help you begin troubleshooting potential problems Raw Data Plot The Raw Data Plot displays the raw reporter fluorescence signal not normalized for the selected wells during each cycle of the real time PCR What to look
88. plates and Low Density Arrays onto the stacks of the Zymark Twister Microplate Handler see page 4 41 4 Run the instrument see page 4 43 5 Analyze the run data Allelic Discrimination Analysis See page 5 5 Absolute Quantification Analysis See page 6 5 Relative Quantification Analysis see page 6 15 Dissociation Curve Analysis See page 6 37 Logging into the 1 If running the SDS software do one of the following Automation Select Instrument gt Disconnect to discontinue communication between Controller the software and the instrument or Software e Select File gt Exit to close the SDS software 2 Start the Automation Controller Software select g stat gt Programs gt amp Applied Biosystems gt amp SDS 2 2 1 gt F Automation Controller 2 2 3 In the Login dialog box enter your user name and password then click x to log into the database 4 Specify the Session Study associations as described in Associating Session and Study Data on page 4 33 5 Prepare and load the plates onto the Plate Handler as explained in Loading Plates onto the Plate Handler on page 4 41 4 32 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Operating the Software with an SDS Enterprise Database Associating User Access Requirement EA If using the SDS Enterprise Database all users can associate
89. prevents the instrument from uploading the firmware from the computer and causes the software to display an error Reading the The 7900HT instrument contains three lights located on the lower left side of the Instrument front panel to indicate the status of the instrument see Figure 2 2 Status Lights Status lights Figure 2 2 Status Lights of the 7900HT Instrument Table 2 1 Instrument Status Lights Power button Light Appearance Status Action Solid The 7900HT instrument is on and in idle state ready to run Ed Flashing Green e Interlocks are open and or the scan head has not reached the safe position e The instrument door is open None This state indicates normal instrument function Flashing i Orange ge Solid The 7900HT instrument is transmitting receiving data to from the computer usually during a run If the light remains on during startup for more than 2 min e The instrument did not boot properly or e 7900HT instrument has experienced a system failure None This state indicates normal instrument function 1 Check that the computer is powered on and connected to the instrument See page 1 10 for a diagram of instrument connections 2 If so power off the instrument wait for 30 sec and then restart as explained on page 2 4 Ap
90. puaser 3 Practice resizing the wells of the plate grid until you are comfortable using the feature 2 26 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Basic Software Skills Tutorial Lesson 4 Using the Hand Held Bar Code Reader Overview The hand held bar code reader functions as an extension of the keyboard that you can use to automatically enter bar codes into the SDS software When the reader is used successfully to scan a bar code it automatically e Transmits the alphanumeric equivalent of the bar code to the software The software enters the bar code text wherever the cursor is active e Transmits a carriage return the equivalent of pressing the Enter key Exercise The following procedure explains how to enter a bar code number using the had held Entering Bar bar code scanner Normally you would scan the bar code into the New Document Code Information dialog box during plate document creation Using the 4 Hand Held Bar Code Reader 2 3 Select Tools gt Document Information In the Document Information dialog box click the Comments field Click here While holding the hand held bar code reader 20 to 30 cm away from a plate aim at the center of the bar code and press the trigger The scanner emits a sweeping laser beam that appears as a red line on the plate Slowly move the scanning beam slowly across the bar code until the scan gun emits a high pitched t
91. quantification The 7900HT instrument collects fluorescence data during each cycle of a pre programmed PCR run The pre programming occurs by setting the thermal cycling parameters of a default method The step parameters time and temperature are adjusted hold cycle set or step are selected and optionally auto increment values ramp rate values and data collection options can be configured A process by which the quantity of a nucleic acid target relative to a control target in one sample is calculated relative to the quantity in another sample For example the quantity of c myc in a brain tissue sample relative to the quantity of c myc in liver tissue sample Fluorescent dye used to bind to the 5 end of the TaqMan Probe There are a number of dyes such as FAM and VIC that covalently bind to the 5 end of the probe Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide run session single nucleotide polymorphism SNP singleplex SNP spectral bin spectral calibration spectral crosstalk stack study task Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide The normalized reporter signal is the cycle by cycle ratio of the fluorescence of the reporter dye and the fluorescence of the passive reference dye in a given well During PCR R increases as the amplification copy number increases until the reaction approaches a plate
92. run click Fost Read The instrument tray rotates to the IN position and the instrument performs the run or plate read Note Before starting the run the instrument may pause up to 15 minutes to heat the heated cover to the appropriate temperature Note For more information on the elements of the Real Time and Plate Read tabs click 2 and see the Sequence Detection Systems Software Online Help The following options are available during and after the completion of the run To See Page Monitor the progress of the run 4 27 Stop the run 4 28 IMPORTANT If you must stop a run in progress for any reason carefully read the instructions on page 4 28 before halting the run Open the instrument tray after the run 4 29 Analyze the run data after the run is complete 4 29 4 26 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Monitoring Instrument Progress Running a Single Plate Using the SDS Software The SDS software displays instrument status and run progress in the Real Time tab real time runs or the Plate Read tab end point runs of the respective plate document Figure 4 10 shows examples of the tabs during operation of the 7900HT instrument Real Time queue Plate Read queue 9 9 E Pre Read 2 Stop Disconnect OnensClose Stop Connect Open Close Post Read Status Method Running Time Remaining 00 01 Status Method Running T
93. section must be equal to the number defined in the Number of Markers section see number 7 above e Leave the markers list line blank in setup files generated for relative and absolute quantification Real time plate documents do not use markers Format fora marker name lt tab gt alleleX lt tab gt alleleY lt tab gt description lt tab gt single marker comments lt cr gt Examples Example Code A 5 Allelic discrimination setup table file SDS Setup File Version 3 Output Plate Size 384 Output Plate ID 384N75822034 Number of Detectors 2 Detector Reporter Quencher Description Comments CYP 2C9 2 1 FAM PDAR CYP 2C9 2 All Example Probe CYP 2C9 2 lt 2 VIC PDAR CYP 2C9 2 Al2 Example Probe xxx Number of Markers 1 Marker AllelexX Alleley Description Comments CYP 2C9 2 CYP 2C9 2 2 CYP 2C9 2 2 PDAR CYP 2C9 2 Example marker Example Code A 6 Absolute or relative quantification setup table file SDS Setup File Version 3 Output Plate Size 96 xxx Output Plate ID 960G729FF9I03 Number of Detectors 3 Detector Reporter Quencher Description Comments GAPDH VIC GAPDH Probe Example Probe SYBR Green SYBR SYBR Green I Example Probe RNase P FAM TAMRA RNase P Probe Example Probe Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 9 Appendix A Software Reference Well Detector Information Element numbers 10 and 11 define the contents of the wells on the
94. select the NTC cluster The SDS software highlights the datapoints within the allele plot and the plate grid b In the plate grid check the wells containing Unknown samples for selected wells that clustered with the NTCs Samples that clustered with the No Template Control wells may e Contain no DNA e Contain PCR inhibitors e Be homozygous for a sequence deletion Setup Instrument J a Legend Allele X Allele Y Allele X amp Y NTC X Undetermined Allele Y CYP 2C19 2 1 NTC cluster selected No Template Control wells FDI III IID Unknown samples clustered with the NTCs 4 Retest any samples that did not cluster tightly or clustered with NTCs to confirm the results 5 If evaluating for multiple markers do the following a In the Marker drop down list select a different marker b Repeat steps 1 through 4 for the new marker c Repeat steps a and b until the calls for each marker have been verified Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 5 17 Chapter 5 Analyzing End Point Data After the Analysis User Access Requirement Post Analysis Options Changing the Display Settings Printing a Report Exporting Plate Document Data If using the SDS Enterprise Database you must belong to the Scientist or Administrator User Group to save the results of an analysis to the database The following options a
95. ses A 16 Exporting Plate Document Data 0 00 ccc eens A 16 Operating the SDS Software from a Command Line 0000 00 eee A 18 Usma INE search TOO avidin chk Se ee Reed Bee es eRe Bed ee Gls ak A 20 Connecting SDS Software to the Database 2 00 00 c eee ee A 22 Designing TaqMan Reagent Based Assays Assay Development Guidelines 0 0 00 cc ee eee eee B 2 Design Tips for Allelic Discrimination Assays 0 000 cece eee ee eee eee B 5 Design Tips for Quantitative PCR Assays 0 0 0 ccc eee B 6 Kits Reagents and Consumables Interchangeable Sample Block Modules and Accessories 0000000008 C 2 Consumables and Disposables 0 000 ccc ee eens C 3 Instrument Maintenance and Verification 0 0 0 ce eee C 5 TaqMan Pre Developed Assays and Reagents 00 cece eee eee eee C 6 Custom Oligonucleotide Synthesis 2 00 0 cece eens C 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Appendix D Theory of Operation Fluorescent Based Chemistries Real Time Data Analysis 0 00 0 ce eee ees Comparative C Method of Relative Quantification Appendix E Limited Warranty Statement Bibliography Glossary Index Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Preface
96. setting Background is too high See below gt 2500 FSU Background is too high Sample block 1 Construct and run a new gt 2500 contamination background plate 2 See Isolating Sample Block Contamination below Background plate contamination aFluorescent standard units The measure of amplitude displayed along the Y axis of the Background Plot Isolating Signals exceeding 2500 FSU are considered outside the limit of normal background Sample Block fluorescence and indicates that the either the background plate or the sample block Contamination module may be contaminated 1 Ifnot already open open the plate document for the background run 2 In the toolbar click f Hide Show System Raw Data Pane The SDS software displays the raw data pane for the background run Select all wells in the plate document 4 Inspect the raw background data for an aberrant spectral peak or peaks Wells producing raw spectra that exceed 2500 FSU are considered irregular and could be contaminated The following figure illustrates the raw data produced by a run on a sample block module containing a contaminated well Amplitude Possible contamination 1 00 E a 10 20 30 Wavelength Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 8 9 Chapter 8 Troubleshooting 5 Identify the location s of the contaminated well s on the sample block by selecting inc
97. the Comparative C Method of data analysis The Comparative C Method is similar to the Standard Curve Method except that it uses arithmetic formulas to achieve the same results for relative quantification Instead of a standard curve the Comparative Cr Method calculates relative gene expression using the equation Relative Quantity 24 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 3 Chapter 6 Analyzing Real Time Data 6 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 6 1 In This Section Section 6 1 Absolute Quantification Absolute Quantification This section contains the following information Oo ETONE babe ee ek eho E EE 6 6 Pe A bas weer eha sos eens ood es 64 eee EAEE 6 8 Analysis tO erpe 0 oe ao 044 6 bo os ee ee Oe 6 9 Pe he Ps 6 oo ho 6 hh eh EEE EASE R ELS OG HARSH 6 10 PAA VS I sh 4 BR ELSE SEER ED OERER EO OHS ESTEE CREO OS 6 11 Viewing nce 3 eee Seb sane dhs os o0ane 4d Grebe sere seeers 6 13 eae Ts a os oe a 9 E E EEE EE eek es eee eee es 6 14 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 5 Chapter 6 Analyzing Real Time Data Overview About Absolute Quantification Employing the 5 Nuclease Assay The Applied Biosystems 7900HT Fast Real Time PCR System supports real time absolute quantification of nucleic acids using a standard curve method The objective o
98. the new stage In the Thermal Profile tab click Add Dissociation stage The SDS software inserts a temperature ramp at the end of the thermal profile consisting of a set of default steps You can customize the default temperature ramp however Applied Biosystems recommends that you observe the following guidelines to ensure the maximum resolution for the run the greatest separation of the derivative peaks during the analysis Guideline Example The Start and End steps of Thermal Profile the temperature ramp must e be separated by a minimum temperature difference of 35 C e elapse 15 seconds 0 15 each End step Start step The ramp rate setting for the Ramp Rate End step of the temperature Stage 4 ramp must be 2 IMPORTANT You must ae sia End deselect the 9600 Emulation nd step check box to modify the ramp rate D 60 0 setting Ramp Rate Settings xl I eee Data Colection thermal profile must contain Stage 4 a data collection icon Daia 2 si collection i icon 0 60 0 xl I 5 Go to Step 6 Saving the Plate Document as a Template on page 3 22 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 21 Chapter 3 Preparing a Run Step 6 Saving the Plate Document as a Template User Access Requirement Adjusting the Display Settings 3 22 Optional If using the SDS Enterprise Da
99. the stack by adjusting the Rotary Adjustment arrows as needed After the Plate Handler arm is centered inside the stack click FinaPlate The Plate Handler arm lowers upon the plate Confirm the following e The plate is in the middle of the gripper span e The plate sensor switch is contacting the plate e The gripper does not contact the side of the stack Click Close Gripper Click ertical Home The Plate Handler arm raises to its highest position If the plate contacts the sides of the stack re adjust the rotary position of the Plate Handler arm until the plate moves freely in the stack Note Contact between the plate and the stack may be unavoidable However try to minimize the contact as much as possible Using the Vertical Positioning commands raise and lower Plate Handler arm several times to check the alignment Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 47 Chapter 7 Maintaining the Instrument 10 11 PA 13 Click Ya Rotary Offset and click s The software records the rotary position for the Zymark position 5 input stack 2 Repeat steps 1 through 9 for input stacks 3 and 4 to define Rotary Offset values for the remaining positions 6 and 7 Zymark position 7 input stack 4 Zymark position 6 input stack 3 Exit the Zymark Twister Software a Click ManMenu b Click Exit The software closes A bug in th
100. the three possible genotypes allele X homozygous allele Y homozygous and heterozygous Because of their homogenous genetic compliment homozygous samples exhibit increased fluorescence along one axis of the plot depending on the allele they contain In contrast heterozygous samples appear within the center of the plot because they contain copies of both alleles and therefore exhibit increased fluorescence for both reporter dyes About Outliers Samples that did not cluster tightly may e Contain rare sequence variations e Contain sequence duplications e Not contain a crucial reagent for amplification the result of a pipetting error 5 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Before You Begin Before You Begin Using the SDS For specific instructions on any procedure described within this section refer to the Software Sequence Detection Systems Software Online Help To get help at any time during the Online Help procedure click 2 located within the dialog box or window in which you are working Examples in The illustrations and screenshots that appear within this chapter were created from a This Section plate containing Pre Developed TaqMan Assays and Reagents for Allelic Discrimination run to screen 6 human genomic DNA samples HD 1 2 3 4 7 8 for 2 targets CYP 2C9 2 and CYP 2C19 2 Each well of the plate contains 1 uL DNA TaqMan 2X Universal PCR Master Mix forward
101. the well from the analysis but does not delete the data 1 While pressing and holding the Ctrl key select the well s in the grid that you want to remove 2 In the well inspector of the Setup tab click the Omit Well s check box Selected wells removed from use Omit Well s check box selected 3 Program the method for the run as explained on page 3 17 3 16 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Step 5 Programming the Plate Document Method Step 5 Programming the Plate Document Method User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to modify the method of a plate document About SDS During a run the SDS software controls the instrument based on the instructions Methods encoded in the method of the plate document Each new plate document except allelic discrimination contains a default method that you must customize for your experiment Methods contain the e Thermal Cycler Conditions e Auto Increment Values e Ramp Rates e Data Collection Options e Reaction Volume Setting To create a method for Then e absolute quantification program the method as explained on page 3 19 e relative quantification IMPORTANT If using the SDS Enterprise Database read the discussion of considerations below allelic discrimination see page 3 18 dissociation curve analysis T
102. tne eS ES BarCode Readers riser uraan ie OE Bh SS OA Ee BA ESS Zymark Twister Microplate Handler 0 0 0 c eee ee eee Compatible Consumables 000 cee ee eee eee INSULUMENTIG ONNECIONS csi pn canda bind TANA E a Awe a Ga eek er Section 1 2 Getting to Know the Software 0 0000 eee ee eee SOftWare COMPONEINS cas ctacerdcsateke meas tes ates Grae ae eee ees SDS Software Related Files and Formats 2 00000 eee eee eee Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide iii Chapter 2 Chapter 3 Managing Sequence Detection System Data 00 ees 1 17 Section 1 3 SDS Enterprise Database 0 cc ee 1 21 About the SDS Enterprise Database Feature 0 0 ccc es 1 22 About the SDS Enterprise Database Software Suite 0 0 0 0 2 eee 1 25 Database Modules for Large Scale Analysis 00 0c eee eee eee 1 25 Database Management Utilities 0 0 0 ee 1 26 Database Design and Information Management 0000 cece eee eae 1 27 Supporting API Documentation 0 0 0 ccc ee tee eee 1 28 Getting Started geting SLANG ars easy aae a eevee cn eas a Ya Se Sve eE ee a hse EO Soe ee ae 2 2 Powering On the 7900HT Instrument 0 0 0 ee ee 2 4 Using the SDS Software 2 ccc ee ee ee ee eee eee eee eee 2 7 Basic Sortware SKIS TUONA leanna ceed awa nnds baa aw seewhaw beau heeds 2 10 Notes for Database
103. tray and runs the plate according to the parameters defined by the plate document file Note Before starting a real time run the instrument may pause up to 15 minutes to heat the heated cover to the appropriate temperature If using the SDS Enterprise Database the Automation Controller Software automatically conducts a primary analysis of each completed run and saves the results to the database If you specified a study or studies as explained in Associating Session and Study Data on page 4 33 then the software automatically attaches the analyzed session data to the appropriate study The following options are available during and after the completion of the run To See Page Monitor the progress of the run 4 44 Stop the run 4 44 IMPORTANT If you must stop a run in progress for any reason carefully read the instructions on page 4 28 before halting the run Open the instrument tray after the run 4 44 Analyze the run data after the run is complete 4 44 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 43 Chapter 4 Operating the Instrument Monitoring Instrument Progress Stopping the Instrument Ejecting a Plate After the Run Analyzing the Run Data The Automation Controller Software displays the progress of the current run in the Thermal Status tab See page 4 27 for an explanation of the Thermal Status tab To stop the plate queue
104. via the vent port 6 To transfer the sample specific PCR reaction mix from the fill reservoirs into the reaction wells the Low Density Array must be centrifuged Continue with Centrifuging the TaqMan Low Density Arrays on page 4 16 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 15 Chapter 4 Operating the Instrument Centrifuging the TaqMan Low Density Arrays Equipment and You need the following equipment and materials to centrifuge the Low Density Materials Needed Arrays e TaqMan Low Density Array s containing sample specific PCR reaction mix page 4 14 e Sorvall Legend T Centrifuge e Sorvall Heraeus Custom Buckets four required each with its own card holder e Blank balance cards Placing Cards in Note The centrifuge holds four Sorvall Heraeus buckets Each bucket holds up to the Buckets three Low Density Arrays loaded and or blank balance cards 1 Obtain an empty Sorvall Heraeus Custom Bucket and a card holder for the centrifuge IMPORTANT The Sorvall Heraeus buckets and card holders required for the Low Density Arrays are custom made Do not use any other bucket card holder system for this procedure 2 Place the bucket on a lab bench with the label facing you 3 Insert Low Density Arrays into the card holder making sure that e The fill reservoirs project upwards out of the card holder e The reaction wells face the same direction as the Th
105. workflow made up of existing data management solutions The SDS Enterprise Database provides a multi level user security system that restricts access to the database and to most functions of the SDS software and 7900HT instrument The database provides the SDS User Account Manager software with the client version of the SDS software for managing user accounts The User Manager allows users with Administrator privileges to assign privileges and passwords for user accounts and to add and remove them This guide indicates the access privileges required to perform each task using a short note above each procedure Note See the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide PN 4351669 for a complete description of user accounts for the SDS Enterprise Database The SDS Enterprise Database is a relational Oracle based repository designed to store SDS data produced by Applied Biosystems 7900HT Fast Real Time PCR System instruments In addition to serving as a common repository for SDS data the database provides user authentication robust and scalable data management and flexible archive capabilities The database also provides the infrastructure and tools necessary to integrate a 7900HT instrument into a laboratory workflow made up of existing data management solutions The SDS Enterprise Database supports a basic two tier server client architecture within an Ethernet 10Base T or Fast E
106. 03 22 38 25 PST 2002 POAR CYP 2C9 ollele 1 FAM Non Fluorescent i Sun Noy 03 20 52 03 PST 2002 Thu Grek 24 17 24 45 POT 2002 CYP 2C9 Allele 2 VIC Non Fluorescent i Sun Noy 03 22 56 54 PST 2002 Thu Grek 24 17 25 54 POT 2002 FAM Non Fluorescent ee Sun Mov 03 20 38 24 PST 2002 Sun Now 03 20 38 44 PST 2002 CYP 2019 Allele 1 FAM Non Fluorescent IPOAR CYP 2019 Allele 2 VIC Non Fluorescent Cony ho Plate Document Done Repeat steps 2 through 4 to create detectors for all remaining assays on the plate Note Click the 7 button for information on the Detector Manager dialog box or to view the procedures for editing deleting or searching for detectors 6 Choose from the following If constructing a Then plate document for C e absolute quantification copy the detector s to the plate document as e relative quantification explained in the procedure on page 3 11 e dissociation curve analysis allelic discrimination create and apply markers to the plate document as explained on page 3 12 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Step 2 Applying Detectors and Markers Copying IMPORTANT Once you copy a detector to the plate document it is no longer linked and Applying to the corresponding entry in the Detector Manager Consequently if you modify a Detectors to the detector using the Detector Manager after you have copied it to a p
107. 055 7 36 Adjusting the Sensitivity of the Plate Sensor Switch 2000 02 ee eeee 7 37 Aligning the Plate Handler 0 0 0 0 ccc ee eee 7 41 Aligning the Fixed Position Bar Code Reader 0000 e eee eee eee 7 49 Cleaning and Replacing Gripper Finger Pads 0000 cee eee ees 7 52 Section 7 3 Maintaining the Computer and Software 0 00005 7 53 General Computer Maintenance 0 0 cc ee ees 1 54 Maintaining the SDS software 0 0 cece eens 7 55 Chapter 8 Troubleshooting MounleSnhoounGg lable e042 cee A a Ce ee eea Ree eRe ewes 8 2 Low Precision or Irreproducibility 2 0 0 0 cc ees 8 5 BACKOrOUNA a0 6 ee a ce ee ee ee ee 8 9 Pure DYS RUNS nc cay poem ties CO MOOR SP Rae hed eee ete bo eaue tA 8 11 Real Time Runs Quantitative PCR and Dissociation Curves 0 8 12 End Point Runs Allelic Discrimination 0 0 000 eee ee eee 8 13 Software and 7900HT Instrument 0 00 0 eee 8 14 Zymark Twister Microplate Handler and Fixed Position Bar Code Reader 8 17 TaqMan Low Density Array 22248 iitiet tactic ie tae hw keh bee ee ad 8 18 SDS Enterprise Database 0 0 ccc eee ees 8 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide vii Appendix A Appendix B Appendix C vill Software Reference Importing Plate Document Setup Table Files 0 0c ee eee A 2 SElUD SADIE FES eruera e
108. 0HT Fast Real Time PCR System follows during a real time or end point sequence detection run Methods used by the 7900HT instrument make use of the following information thermal cycling times and temperatures ramp rates auto increment values and data collection settings Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide minor groove binder MGB multicomponenting multi reporter multiplex PCR No Template Control NTC normalized reporter signal R nucleotide outlier PCR master mix persist ed pure dye Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide One of a class of molecules which form close atomic contacts in the deep narrow space formed between the two phosphate sugar backbones in the DNA double helix Conjugation of a minor groove binder to oligonucleotides stabilizes nucleic acid duplexes causing a dramatic increase in oligonucleotide T Fluorogenic probes with the MGB attached to the 3 end perform well in the 5 nuclease assay They are an improvement over unmodified probes because shorter sequences 13 to 20 mers can be used to obtain probes that have an optimal T Thus attachment of the MGB enables the use of shorter fluorogenic probes which results in improved mismatch discrimination The analysis performed by the SDS software used to distinguish the contribution of individual dyes to the raw spectral dat
109. 1521 You may need to check with your database administrator if the port setting has been modified e Service Name The Service Name setting is configured during the installation of the SDS Enterprise Database The default is SDS22 The SDS software allows users of the Administrator User Group to disable the database functions of the software When disabled the software hides the toolbar buttons and menu commands for opening and saving data to the database Note The enable disable feature can be used during periods when the network connection is unavailable By disabling the database connection the SDS software does not require users to log in before using the 7900HT instrument After the database becomes available again the data collected in the interim can be uploaded to the SDS Enterprise Database using the SDS Document Loader For more information see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide P N 4351669 To disable or enable the database features of the SDS software IMPORTANT You must belong to the Administrators User Group to modify the database options 1 Start the SDS software see page 2 7 for more information 2 In the Login dialog box enter a user name and password of a user account that belongs to the Administrator user group see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide for more
110. 2 1999 Sangoram A M et al Mammalian circadian autoregulatory loop a timeless ortholog and mPer1 interact and negatively regulate CLOCK BMAL 1 induced transcription Neuron 21 1998 1101 1113 Sgroi D C S Teng G Robinson R LeVangie J R Hudson Jr and A G Elkahloun 1999 In vivo gene expression profile analysis of human breast cancer progression Cancer Res 59 5656 5661 Shayesteh L Lu Y Kuo W L Baldocchi R Godfrey T Collins C Pinkel D Powell B Mills G B and Gray J W PIK3CA is implicated as an oncogene in ovarian cancer see comments Nat Genet 21 1 99 102 1999 Shimokawa T et al Transcriptional regulation of muscle specific genes during myoblast differentiation Biochem Biophys Res Commun 246 1998 287 292 Taoufik Y Froger D Benoliel S Wallon C Dussaix E Delfraissy J F and Lantz O Quantitative ELISA polymerase chain reaction at saturation using homologous internal DNA standards and chemiluminescence revelation Eur Cytokine Netw 9 2 197 204 1998 Bibliography 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Virtanen M H Torma and A Vahlquist 2000 Keratin 4 upregulation by retinoic acid In vivo A sensitive marker for retinoid bioactivity in human epidermis J Invest Dermatol 2000 Mar 114 3 487 93 Wingo S T Ringel M D Anderson J S Patel A D Lukes Y D Dyuh Y Y Solomon B Nich
111. 2 value only for the standards that make up the curve e Standard curve plot A scatterplot of datapoints from the absolute quantification run Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 13 Chapter 6 Analyzing Real Time Data Viewing the Displays the results of the absolute quantification calculation in the results table of Analysis Table the plate document Figure 6 6 shows an example of the results table containing the data from an RNase P Instrument Verification Plate Posit Sample Detector Task ct Quantity Gtymean Otystddev Help button Al Unknown Po RMase P Unknown 2465946 4641 366 4905 1187 478 2128 AZ Unknown Po RNase P Unknown 24 617865 4774 904 4905 1187 478 2128 ee E AS Unknown Po RNase P Unknown 24 523537 5091 7935 49051187 479 2128 Figure 6 6 Analysis Table of the Absolute Quantification Plate Document The columns of the table are e Position The coordinate position of the well on the plate e Sample The sample name applied to the well e Detector The name of the detector assigned to the well e Task The task NTC Standard or Unknown assigned to the well Note See page 3 14 for information on applying tasks to the plate document e Cr The threshold cycle generated by the well during the PCR Note The software displays Undetermined in the Cy column when the associated sample fails to p
112. 4 10 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Preparing TaqMan Low Density Arrays for Use Internal Structure Figure 4 6 illustrates the Low Density Array internal structure Fill port Vent port Fill reservoir O O Main Feeder channel Reaction well Figure 4 6 TaqMan Low Density Array Internal Structure Detectors The Low Density Array permits the amplification of target and an external endogenous control cDNA using fluorogenic 5 nuclease assays The assays consist of two reactions each a complete PCR system with corresponding probe and primers The fluorogenic probes of the assays function as follows e Probes labeled with the FAM dye detect the amplification of user specified cDNA targets e Probes labeled with the FAM dye detect the amplification of the external endogenous control Genomic DNA TaqMan probes and primers for the cDNA targets span exon exon junctions to Contamination minimize the contribution of contaminating genomic DNA Performance tests demonstrate that 5 exonuclease assays can be run with samples containing up to 10 000 copies of genomic DNA without detection of contaminants unless otherwise specified by Applied Biosystems External Endogenous Control Assay Note that the commonly used external endogenous control assay is not RNA specific and consequently is affected by genomic DNA contamination However because of the extremely hig
113. 43 5 Analyze the run data Allelic Discrimination Analysis see page 5 5 Absolute Quantification Analysis see page 6 5 Relative Quantification Analysis See page 6 15 Dissociation Curve Analysis See page 6 37 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Operating the Software without an SDS Enterprise Database Adding a Plate Document to the Plate Queue Using the SDS Software IMPORTANT A plate document must contain a bar code before you can add it to the plate queue See page 3 25 for more information on configuring a plate document with bar code information 1 Inthe SDS software select the Instrument tab 2 In the Instrument tab select the Queue tab 3 In the Queue tab click sendtoqueve Have you saved the plate document Yes then the software automatically saves the plate document No do the following a In the Look in field of Save As dialog box navigate to a directory where the software can save the new file b Select Files of type gt SDS 7900HT Document sds c Click the File name field then do one of the following Enter a name for the plate document file Enter or scan the bar code number of the plate into the field d Click _ sve The software saves the plate document 4 When prompted click ves to submit the document to the plate queue After you have added a plate document to the plate queue the software lo
114. 6 SK RNaseP nknown 4 gt _JoOmit wellis Table pane page 2 29 Figure 2 8 Common Elements of SDS Plate Documents page 2 30 Well inspector page 2 31 The cells of the plate grid correspond to the wells of the reaction device that the plate document models The grid displays well information according to the type of plate document The Plate Grid properties settings in the Display Settings dialog box determine content and appearance of the information displayed inside the cells of the grid Note For more information on configuring the display settings for the plate grid click 2 help button Table Pane The table pane displays the setup and analysis properties for the plate document You can export the table pane as a tab delimited text file for use by a spreadsheet application see Appendix A Software Reference for more information Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 29 Chapter 2 Getting Started Tabs below Table 2 5 Tabs of the SDS Software Plate documents can have up to six tabs depending on their function as described Tab Function Views Plots Setup Displays well information and allows you to configure the plate grid with setup information Well Inspector Instrument Used to program the plate document method run the plate document or send it to the Plate Queue e Method Editor Thermal Profile ta
115. 900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 6 2 Relative Quantification Section 6 2 Relative Quantification IV This Section OVerviOW cise o4 64440 404455n0ees4ene anes Er 4545 PARA Eee ee ees 6 16 Essential Experimental Components 0 0 00 e eee cece eens 6 20 ig ih ee een Ga dk oe EA oS S49 RDA S ES ETE CEER E 6 21 Analysis Checklist 0 0 0 0 000 0c eee eens 6 22 Options for Analyzing Relative Quantification Data 04 6 23 Ce Ts oa ye kon oe ee RSE ES ERE HES HES ODOR OEE EO 6 23 Analyzing the UY 645 3k xb 4 94 60 4h 00 itir EEN r RANNAR 6 26 baaada li T E eee o aes sees a4 dees Pee ese ess TET 6 31 Amer Ps as 04 oo es 0 9 9 0 oe oe ee ees 6 35 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 15 Chapter 6 Analyzing Real Time Data Overview Relative Quantification on the 7900HT Instrument Employing the 5 Nuclease Assay The Applied Biosystems 7900HT Fast Real Time PCR System supports real time relative quantification of nucleic acids using the Comparative C method The Comparative Cy Method uses the following arithmetic formula to achieve results for relative quantification 7 AAC where AAC The normalized signal level in a sample relative to the normalized signal level in the corresponding calibrator sample Note The derivation of the above equation is explained in ABI PRISM 7700 Sequence Detection
116. 98 744 746 Luthra R et al Novel 5 exonuclease based real time PCR assay for the detection of t 14 18 q32 q21 in patients with follicular lymphoma Am J Pathol 153 1998 63 68 Maudru T and K W Peden Adaptation of the fluorogenic 5 nuclease chemistry to a PCR based reverse transcriptase assay BioTechniques 25 1998 972 975 Nagai M Usuku K Matsumoto W Kodama D Takenouchi N Moritoyo T Hashiguchi S Ichinose M Bangham C R Izumo S and Osame M Analysis of HTLV I proviral load in 202 HAM TSP patients and 243 asymptomatic HTLV I carriers high proviral load strongly predisposes to HAM TSP J Neurovirol 4 6 586 593 1998 Nogva H K and Lillehaug D Detection and quantification of Salmonella in pure cultures using 5 nuclease polymerase chain reaction In Process Citation Int J Food Microbiol 51 2 3 191 196 1999 Pusterla N Huder J B Leutenegger C M Braun U Madigan J E and Lutz H Quantitative real time PCR for detection of members of the Ehrlichia phagocytophila genogroup in host animals and Ixodes ricinus ticks J Clin Microbiol 37 5 1329 1331 1999 Pusterla N Leutenegger C M Chae J S Lutz H Kimsey R B Dumler J S and Madigan J E Quantitative Evaluation of Ehrlichial Burden in Horses after Experimental Transmission of Human Granulocytic Ehrlichia Agent by Intravenous Inoculation with Infected Leukocytes and by Infected Ticks J Clin Microbiol 37
117. Automation Controller Software cannot find a plate document file Computer and or software displays the Run Completed Successfully dialog box but will not respond and appears to be frozen Run will not start Computer is slow when analyzing data opening or closing dialog boxes and other software processes The computer will not logon to the Windows Operating System The computer will not boot up at all Qo N Zymark Twister Microplate Handler and Fixed Position Bar Code Reader Plate Handler emits grinding noise when picking up or putting down plates Plate Handler arm contacts racks when retrieving or stacking plates Plate Handler arm releases plates awkwardly into the plate stack Reaction plates tip or tilt when placed into the instrument tray by the Plate Handler arm Plate Handler fails to sense or grasp plates Plates stick to the gripper fingers of the Plate Handler arm Plate Handler does not restack plates in original locations Fixed position bar code reader not reading plate bar codes Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 8 3 Chapter 8 Troubleshooting Table 8 1 Troubleshooting Table Category Symptom Page TaqMan Low Density After removing the Low Density Array from its 8 18 Array packaging the fill consumable is damaged creased bent or folded After removing the Low Density Array from its packaging
118. Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Basic Software Skills Tutorial Exercise 1 You will need to create a plate document for every plate you run on the 7900HT Creating a Plate instrument Document IMPORTANT The SDS software can handle multiple documents simultaneously however the processing speed of your computer will decrease with each open document For that reason Applied Biosystems recommends limiting the number of open documents to no more than 10 1 Ifnot already active start the SDS software a Select g amp stat gt Programs gt amp Applied Biosystems gt amp SDS 2 2 1 gt ESDS 2 2 1 b Database Only In the Login dialog box enter your user name and password and click xl User Mame Passyvord Database Setup Cancel 2 Click or select File gt New 3 Configure the New Document dialog box with the following settings e Assay drop down list Select Absolute Quantification e Container drop down list Select 96 Wells Clear Plate e Template drop down list Select Blank Template e Barcode field Enter Practice Note Normally you would enter a file name or bar code consisting of up to 128 characters in the Barcode field However for the purpose of demonstrating the software function this tutorial instructs you to enter Practice Assay Absolute Quantification Container 384 Wells ClearPlate sss Template
119. Biosystems 7900HT Fast Real Time PCR System has an installation Rating overvoltage category of II and is classified as portable equipment Physical Hazard Safety v Moving Parts gt PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing the instrument AN DOl PHYSICAL INJURY HAZARD Do not operate the instrument without the arm shield in place Keep hands out of the deck area when the instrument is spotting Biological Hazard Safety General ANTEE BIOHAZARD Biological samples such as tissues body fluids Biohazard and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Read and follow the guidelines in these publications e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 http bmbl od nih gov e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Additional information about biohazard guidelines is available at http www cdc gov Laser Safety Laser The Applied Biosystems 7900HT Fast Real Time PCR System uses a Class 3B laser Classification Under
120. C pplied Biosystems D5 Documents C pplied BiosystemsD5 Documents C pplied Biosystemse0D5 Documents File Search Added plate document files E E Local Disk C EL Applied Biosystems i a sps Eroen FeyRG_Plated1 sds Rijko PlateO2 sds RRO Platea ade SA Ra PlateO4 sce i i p H 0 AppledBiosysterms HL BACKUP HL c_DILLA LD Config Mai Humber of Plates Study can hold 10 6 free OK Renee Selected plate document files Existing Plates In The Study If you do not know where your plate documents are located or want to search for specific plate documents a Select the Search tab b In the Search in field enter a directory address to search or click and select a location directory or hard drive to search c If you want the SDS software to search all subdirectories in the chosen location select Include Subdirectories 6 24 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Creating the Study d In the Plate Query dialog box configure the Find Plates Matching These Criteria table with parameters that correspond to the documents of interest e Click _ Search f In the Search Results list press and hold the Ctrl key select the files of interest then click add Plates To Be Added To Study C pplied Biosystems SDS Docume Plate documents C pplied Biosystems SDS Docume C pplied Biosystems SDS Docu
121. D RNaser SK Pooulatian SK Pooulation Sample name n k i Pim E LEL Fanal z a appears here Sir Mer i welis A1 C12 2 r i Sample Mame ak Population Sample Name field 4 Configure the plate document with plate information as explained below Configuring the 1 Inthe SDS software select Tools gt Document Information Plate Document Information Optional In the Document Information dialog box edit the Barcode Operator or Plate Comments information IMPORTANT If using an SDS Enterprise Database e The bar code entered into the plate document must be unique and cannot be used in another plate document e The software automatically populates the Operator field with the name of the user who logged into the SDS software and performed the data collection The software populates the field only after the plate document has been run e Do not modify the bar code of a plate document that contains data from a run 3 When finished click 4 Run the plate document and associated plate as explained on page 3 26 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 25 Chapter 3 Preparing a Run Step 9 Running the Plate on the 7900HT Instrument User Access There is no access requirement All users can run plates that have been saved to the Requirement SDS Enterprise Database IMPORTANT A plate document must be saved to the
122. Dye Run Extracting Pure Dye Information from the Run The purpose of viewing the data in the Pure Dye Wizard is to eliminate irregular pure dye peaks from the data set The wizard presents the spectral data from the pure dye plate or card in sets of three wells each containing the same pure dye Because the wells displayed by the wizard contain the pure dye at an identical concentration the signal peaks for the set should be identical Occasionally pipetting inaccuracies or contamination can cause a well signal to shift slightly While viewing the data the outlying peaks must be eliminated l 2 Select Analysis gt Extract Pure Dye Wizard Follow the instructions as explained by the Extract Pure Dye Wizard to extract the pure dye spectra When presented with each screen do the following a Inspect the spectra for shifts in peak location Example Spectra from a Pure Dye Plate x Pure Dye Wizard Review Row D TET Dye Review the TET dyes in row and omit any dye that does not meet the quality standards Wavelength shift tl Fe Sp EE ay aye S aaa T a a ke S e lee e GILES hf ee T aa aa i aaa a aa e Click here to remove it xi Review Row D TET Dye Review the TET dyes in row and omit any dye that does not meet the quality standards Wavelength shift He eC ey a EE aia SIGE Si e E E Sia e a e ee a a a e he erst ac I Rd ei fy Keep Dye Click here to remove it Applied Biosystems 7900HT Fast R
123. Enterprise Database User Guide 7 21 Chapter 7 Maintaining the Instrument Preparing the Pure Dye Plates or Cards Preparing the IMPORTANT A background run must be performed before running a pure dye plate Pure Dye Plates See Performing a Background Run on page 7 16 for more information l 2 3 Remove the pure dye plate from the refrigerator or freezer and set it aside to warm to room temperature Remove the pure dye plate from its packaging Briefly centrifuge the pure dye plate Preparing the IMPORTANT A background run must be performed before running the microfluidic Microfluidic Pure pure dye cards See Performing a Background Run on page 7 16 for more Dye Cards information l Remove the three empty microfluidic pure dye cards from the refrigerator and set them aside to warm to room temperature Remove the three dye tubes FAM VIC and ROX dyes from the freezer to thaw When the dyes have thawed vortex the tubes Load the FAM dye into one of the empty microfluidic pure dye cards loading 100 uL of dye per fill reservoir Centrifuge the microfluidic pure dye card per the procedures in Centrifuging the TaqMan Low Density Arrays on page 4 16 Seal the microfluidic pure dye card per the procedures in Sealing the TaqMan Low Density Arrays on page 4 19 Repeat steps 3 through 5 for the VIC and ROX dyes Preparing a Plate Document for a Pure Dye Plate or Card Preparinga 1 2 Cli
124. For additional documentation see How to Obtain Services and Support on page xii Send Us Your Applied Biosystems welcomes your comments and suggestions for improving its Comments _ user documents You can e mail your comments to techpubs appliedbiosystems com How to Obtain Services and Support To contact Applied Biosystems Technical Support from North America by telephone call 1 800 899 5858 For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Services and Support At the Services and Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Services and Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities xii Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide safety and EMC Compliance Information This section includes the following topics Safety Conventions Used in This Document 0 00000002 ccc eee XIV Symbols on Instruments a no er ere eer XV safety Labels on Instruments 2 00 62 0505 00 cscs cence eeeeeeteevees XVI
125. GP late Cid veiue New Wale Default descriptions Calculation Error Meed to Change Threshold Enter ihe Feaccy for Change Custom description of the reason for the change Figure 5 1 Reasons for Change Dialog Box Options The Electronic Signature Verification dialog box appears only if the SDS Enterprise Database is configured to require electronic signatures The electronic signature system restricts and tracks user access of the SDS data contained by the database When the Electronic Signature Verification dialog box appears 1 In the User ID and Password fields enter your user name and password then click 2 After the software validates your user name and password it displays a message stating the success or failure of the signature Click x to continue Description of the action that requires a signature Enter your user name Enter your password Figure 5 2 Electronic Signature Verification Dialog Box Options Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 5 3 Chapter 5 Analyzing End Point Data 5 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 5 1 Allelic Discrimination Section 5 1 Allelic Discrimination in This Sechon VCR Wy co oo on che nts 1659 40944950458 2480059 ER EEE eens 5 6 Pere T CG oy on daeo ee wees REAN ERRARE ETENEE 5 9 Pare eo op 5 5h hh og 2 a es ee wh
126. General Toolbar for data management and analysis The following list summarizes the functions of the tools and provides a reference in parenthesis to a procedure involving the function O Creates a new plate document see page 2 13 Opens a plate document from the hard drive see page 2 19 E Saves the plate document to the hard drive see page 2 17 Opens a plate document from the database see page 2 19 Saves the plate document to the database see page 2 17 IMPORTANT The general toolbar will display the and J options only if the SDS software is connected to an SDS Enterprise Database Imports data from a text file see page 2 16 1 Exports data to a text file see page 2 14 amp Opens the Print Report dialog box see page 2 13 Opens the Find tool see page 2 13 Removes the selected object and places it into memory see page 2 13 Copies the selected object into memory see page 2 13 Inserts the object in memory into the current selection see page 2 13 Analyzes the plate document see page 2 13 Opens the Analysis Settings dialog box see page 2 13 About the The display toolbar contains tools used to control the display of information inside Display Toolbar the SDS software workspace The following list summarizes the functions of the tools and provides a reference in parenthesis to a procedure involving the function Hides shows the Well I
127. Guide Analyzing End Point Data In This Chapter End Point Runs on the 7900HT Instrument 0 000 00 eae 5 2 Notes for Database Users ooo hos ed Hee eee sd HERO REE OLEH Reh ES RROD 5 3 Section 5 1 Allelic Discrimination ccc cece ccc ccccecceees 5 5 Og og eo eth be ee EE ee eee eee aes 5 6 Were WE gh oa ey T 9 ess 5 9 PI CC ed oe da Sb os oS ee ewe 9408 46 Oe eee 5 10 penis 1e Ron Dias a oe gre oy og ee ON ES Oe EE ee REO RS 5 11 Analyzing an Allelic Discrimination Run 0 0 0 00 00 ccc eee 5 12 Calling and Scrutinizing Allelic Discrimination Data 4 4 5 13 pee es FS bn os 4h 2 hen E E ee a sb be ces 5 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 5 1 Chapter 5 Analyzing End Point Data End Point Runs on the 7900HT Instrument End Point Runs End point is the term used to describe the category of sequence detection runs in 5 2 which the Applied Biosystems 7900HT Fast Real Time PCR System is used to measure the fluorescence of a biological sample after it has undergone thermal cycling Unlike real time runs that can yield quantitative measurements the focus of end point experiments is typically to produce a qualitative result End point analysis is commonly used in combination with TaqMan reagent based chemistry to confirm the presence or absence of specific target nucleic acid sequence in cells tissues or fluid samples
128. HT instrument can be used to determine the absolute or relative quantity of a target nucleic acid sequence in a test sample by analyzing the cycle to cycle change in fluorescence signal as a result of amplification during a PCR This form of quantitative PCR analysis called kinetic analysis was first described using a non sequence specific fluorescent dye ethidium bromide to detect PCR product Higuchi et al 1992 Higuchi et al 1993 The use of TaqMan probes and reagents further enhances the method by providing sequence specific amplification of multiple targets for comparative or relative quantification The fewer cycles it takes to reach a detectable level of fluorescence the greater the initial copy number of the target nucleic acid Amplification Plot S555 S555 SSS 55 S555 S55 5555555555555 5 Phase 3 plateau ae HHHH PHH Phase 2 linear j Phan Rone Hy 44 Phase 1 geometric o NM rie j 2y H i H RAA Bi j 0 0 0 10 20 30 40 Cycle Number Figure D 3 Phases of a Typical Amplification Curve When graphed in real time on a linear scale normal amplification of PCR product generates a curve similar to the one shown in Figure D 3 This amplification curve consists of three distinct regions that characterize the progression of the PCR Phase 1 Geometric Exponential Detection of the high precision geometric phase 1s the key to high precision quantitative PCR The geometric phase is
129. How to Use This Guide Purpose of This Guide Audience Assumptions Text Conventions User Attention Words Safety Alert Words The Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide describes how to prepare maintain and troubleshoot the Applied Biosystems 7900HT Fast Real Time PCR System instrument Zymark Twister Microplate Handler and SDS Enterprise Database This guide is written for technicians scientists and researchers of all skill levels who will use and maintain 7900HT instruments with or without the SDS Enterprise Database This guide assumes that a Applied Biosystems technical representative has installed your 7900HT instrument Plate Handler and SDS Enterprise Database and that you are familiar with e Basic Microsoft Windows operations such as using the mouse choosing commands working with windows and using the hierarchical file system e Electronic storage devices computer hard drives and data files e Assay preparation and basic laboratory techniques This guide uses the following conventions Bold indicates user action For example Enter 0 then press Enter for each of the remaining fields e Italics indicates new or important words and is used for emphasis For example Before performing a pure dye calibration always perform a background run e A right arrow bracket gt separates successive commands you select from a drop down or shortcut menu F
130. Low Density Array contain Applied Biosystems fluorogenic 5 nuclease assays TaqMan reagents see the note below that detect the real time amplification of user specified targets Relative levels of gene expression are determined from the fluorescence data generated during PCR using the Applied Biosystems 7900HT Fast Real Time PCR System Relative Quantification software Note In this document the term TaqMan reagents refers to 5 exonuclease assay reagents that feature Applied Biosystems TaqMan primers and probes includes Applied Biosystems PDARs TaqMan SNP Genotyping and Gene Expression Assays Custom TaqMan SNP Genotyping and Gene Expression Assays and any custom TaqMan reagents The Low Density Array Figure 4 5 acts as a vessel for real time PCR It consists of a series of 384 interconnected wells divided into eight sets of assays Each set provides a user specified number of replicates Each well contains dried Applied Biosystems TaqMan primers and probes for one mRNA target Fill reservoir 1 of 8 A reservoir for the cDNA sample or control before it is centrifugally loaded into the wells Fill consumable The segment of the Low Density Array that contains the eight fill reservoirs After the Low Density Array is centrifuged and sealed the fill consumable is trimmed off Barcode Provides coded access to Low Density Array configuration databases Figure 4 5 The TaqMan Low Density Array
131. Man Low Density Arrays 1 A cycle set point is any temperature desired for a sample block or heated cover 2 In the context of thermal cycling cycle set point could represent one or more temperatures in a step or stage See AC See AAC See AR Glossary 3 detector endogenous control endogenous reference end point plate read run analysis expression fold value hands off automation high throughput HT hold locus marker method Glossary 4 A virtual representation of a gene or allele specific nucleic acid probe reagent used for absolute quantification relative quantification allelic discrimination or other analyses performed on an SDS instrument Examples of reagents represented as detectors include TaqMan probes and the SYBR Green 1 dsDNA Binding Dye Detector for a gene presented at a consistent level in all experimental samples By using an endogenous control as an active reference one can normalize quantification of a messenger RNA mRNA target for differences in the amount of total RNA added to each reaction See endogenous control Refers to processing of samples for allelic discrimination and plus minus scoring chemistries of samples by the 7900HT instrument The 7900HT instrument collects a single reading after prior PCR temperature cycles are completed using a thermal cycler After analysis by the SDS software the resulting multi component data 1s used to assess the presence o
132. NF Q UNKN 0 3 m 96 Sample 12 GAPDH ENDO 0 TNF Q UNKN 0 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 13 Appendix A Software Reference Example Setup Table Files Example Code A 15 Relative quantification setup table document k SDS Setup File Version 3 Output Plate Size 384 Output Plate ID 384JQZ55A4 Number of Detectors 2E Detector Reporter Quencher Description Comments 18S VIC mgb VIC Bcl 2 FAM Human Bcl 2 Bcl xL FAM Human Bcl xL GAPDH FAM Human GAPDH GBP2 FAM guanlylate binding protein 2 GOS2 FAM Human GOS2 protein gene IL 15 FAM Human IL 15 IL 17 FAM Human IL 17 IL 18 FAM Human IL 18 Lina FAM Human IL la IL 1b FAM Human IL 1 beta IL12p40 FAM Human IL 12p40 INF a2 FAM Interferon alpha2 IP 10 FAM Human IP 10 ISG56 FAM Interferon induced protein 56 Nfkb I FAM Nuclear factor of kappa light PKCi FAM Protein kinase C iota RAPIA FAM RAPIA member of RAS oncogene RhoPK2 FAM Homo sapiens Rho associated TNF a FAM Human TNF alpha c fos FAM Human c fos c jun FAM Human c jun c myc FAM Human c myc COL FAM Cancer Osaka thyroid oncogene p53 FAM Human p53 Well Sample Name Detector Task Quantity 1 Al 18S VIC mgb ENDO 0 0 2 Liver 18S VIC mgb ENDO 0 0 GOS2 TARG 0 0 3 Liver 18S VIC mgb ENDO 0 0 Cot TARG 0 0 4 Liver 18S VIC mgb ENDO 0 0 cot TARG 0 0 5 Liver 18S VIC mgb ENDO 0 0 ISG56 TARG 0 0 6 Liver 18S VIC mgb ENDO 0 0 ISG56 TA
133. Plate Container drop down list Template Blank Template Template drop down list Browse Barcode Barcode field El Cancel 4 Click 5 Create and copy detectors and markers to the new plate document as described in Step 2 Applying Detectors and Markers on page 3 9 3 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Step 2 Applying Detectors and Markers Step 2 Applying Detectors and Markers User Access Requirement Creating Detectors If using the SDS Enterprise Database you must belong to the Scientist or Administrator User Group to create and apply detectors and markers Before you can use a plate document to run a plate it must be configured with detector information for the experiment and marker information if performing allelic discrimination A detector is a virtual representation of a TaqMan probe and primer set used for detection of a single target nucleic acid sequence or a primer set utilizing SYBR Green Double Stranded DNA Binding Dye 1 used for the detection of double stranded DNA product Before using the plate document you must create and apply detectors for all assays present on the plate 1 Select Tools gt Detector Manager 2 In the Detector Manager dialog box click e 3 Configure the Add Detector dialog box Description Name field Enter a name for the detector The name of the detector must b
134. Plate Handler gt Twister The Zymark Twister Software starts Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 41 Chapter 7 Maintaining the Instrument 5 Click Manual Control to display the Manual Control dialog box Aligning The alignment of input stack 1 position 4 in the Zymark Twister Software is the Input Stack 1 first step in the alignment procedure This alignment provides the basis for aligning Zymark all subsequent stacks on the Plate Handler Position 4 1 Place an empty plate into input stack 1 Zymark position 4 2 Inthe Zymark Twister Software click position 4 Rotary Positioning Click 4 Rotary Adjustment opa i The Plate Handler arm moves over the input stack 3 Using the Vertical Positioning commands lower the Plate Handler arm until it is just above the stack The Vertical Positioning box offers four ways to move the Plate Handler arm e Move the slider for large increments e Click inside the slider bar to move the arm in 250 step increments e Click the lower arrow on the bar to move the arm in 50 step increments e Click the up or down arrows in the Vertical Adjustment text box to move the arm in step increments e Click the Vertical Adjustment text box enter a value and press Enter to move the arm into a specific location Vertical Positioning Slider Slider bar 250 steps per click Down arrow 50 steps per click Verti
135. Practice 3 Click Search Select Select Enter Barcode Contains Practice E Plate Query x Find Plates Matching These Criteria o variable Condition vauet vaez La arcod O fotas O proe SS E S a A Clear Row Clear All Append Query Results Click to begin the search Search 4 Inthe Search Results list select the Practice plate document and click o Search Results Hssay Type Session ID Operator Absolute Quantification This is an example of apr Click to select the document Open Select All Deselect All Clear All Done Click to open the document 5 Click _ done 6 Goon to Lesson 2 Viewing and Resizing Panes on page 2 21 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Basic Software Skills Tutorial Lesson 2 Viewing and Resizing Panes Overview Because plate documents can display setup and analysis data in multiple views simultaneously the SDS software provides several navigational devices to help manage the information This lesson will teach you to use the different aids to reduce screen clutter and ensure efficient use of the software Exercise 1 You can resize the panes views and plots of plate documents by moving the grey Resizing Panes lines dividing them horizontally and vertically Views and Plots or hi ARE Dividing line click and drag Dividing line click and drag
136. RG 0 0 7 Liver 18S VIC mgb ENDO 0 0 GOS2 TARG 0 0 8 Liver 18S VIC mgb ENDO 0 0 Cot TARG 0 0 9 Liver 18S VIC mgb ENDO 0 0 Cot TARG 0 0 10 Liver 18S VIC mgb ENDO 0 0 ISG56 TARG 0 0 11 Liver 18S VIC mgb ENDO 0 0 ISG56 TARG 0 0 12 Liver 18S VIC mgb ENDO 0 0 GOS2 TARG 0 0 13 Liver 18S VIC mgb ENDO 0 0 Cot TARG 0 0 14 Liver 18S VIC mgb ENDO 0 0 Cot TARG 0 0 15 Liver 18S VIC mgb ENDO 0 0 ISG56 TARG 0 0 16 Liver 18S VIC mgb ENDO 0 0 ISG56 TARG 0 0 381 Liver 18S VIC mgb ENDO 0 0 GOS2 TARG 0 0 382 Liver 18S VIC mgb ENDO 0 0 Cot TARG 0 0 383 Liver 18S VIC mgb ENDO 0 0 cot TARG 0 0 384 Liver 18S VIC mgb ENDO 0 0 ISG56 TARG 0 0 Information for wells 17 through 380 has been removed from this example for brevity A 14 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Example Code A 16 Allelic discrimination setup table document Setup Table Files k SDS Setup File Version 3 ASE Output Plate Size xJ OUCDUt Plate ID xxx Number of Detectors Detector Reporter AL2 1 FAM AL1 1 VIC AL2 2 FAM AL1 2 VIC AL2 3 FAM AL1 3 VIC AL2 4 FAM AL1 4 VIC xxx Number of Markers Marker Allelex SNP1 AL2 1 SNP4 AL2 4 SNP3 AL2 3 SNP2 AL2 2 Well Sample Name 1 Al 2 A2 3 A3 4 A4 5 A5 6 A6 7 A7 8 A8 9 A9 10 A10 11 A11 12 A12 13 A13 382 P22 383 P23 384 P24 Well Sample Name 1 A1 2 A2 3 A3 4 A4 5 A5 6 A6 7 A7 8 A8 9 A9 10 A10 11 A11 12 A12 13 A13
137. RNase Unknown of the well to which the mouse cursor points ed Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 23 Chapter 2 Getting Started Exercise 2 The SDS software provides several methods for selecting wells from the plate grid Selecting Multiple The following exercise will familiarize you with most of them Wells 1 Select a block of wells from the plate grid by doing one of the following e Click and drag the mouse cursor across the block of wells or e Click the well at the top left corner of the block Then while holding down the Shift key click the well at the bottom right corner of the block The software outlines the selected wells with a black border et A ILL i ey Se el lal Sarpi P A e T Toe Deae tate tak a eau Mia FI irira D iL Fibeee Alper AHF Fil sk AHF ail 2 Select several isolated wells of the plate grid by doing of the following a Hold down the Ctrl key and click individual wells to select them The software outlines the selected wells with a black border b While holding down the Ctrl key click the wells to de select them a parum z EE SS ae ale M a wate salto aes h rerrre LLL LE Li w T i wil E p al n BEE a i Sarpi Piara l SESE G wwe pi o e oem oul BREE RESET ee os a iC BEEBE EEE i EEHEEHE AHF AHERE j Fb se 2 24 Applied Biosystem
138. Relative QuanuniCauOn sessa ceased i ereader eee ease oat aeeseehtdade 3 5 RNase P Instrument Performance Verification 0 0 7 30 1 Create an absolute quantification plate document See page 3 8 2 Apply detectors to the plate document a Create detectors for the absolute quantification probes see page 3 9 b Copy the detectors to the plate document See page 3 11 Vv 3 Configure the plate document with tasks and quantities a Configure the plate document with detector tasks NTC Standard and Unknown See page 3 14 b Assign quantities to the wells of the plate document that contain standards see page 3 15 a 4 Program the method for the absolute quantification run see page 3 17 LAA H 5 If performing an assay in which you would like to collect dissociation data add a temperature ramp to the thermal profile to perform a dissociation curve analysis see page 3 21 6 Choose from the following If running a single plate then continue to step 2 f running the first plate in a series of plates with identical assay configurations then save the plate document as a template see page 3 22 Vv 7 Create a plate document from the template created in step 2 see page 3 24 8 Configure the document with sample names and plate information see page 3 25 9 Prepare and run the absolute quantification plate o
139. S plate document file enter com apldbio sds SDSResultsAnalyzerMain e Multicomponent and analyzed data for a plate document file enter com apldbio sds MulticompAndResultsAnalyzerMain 4 Save and close the batch file 5 Invoke the file at the command line by entering SDS RESULTS bat sdsfile sds sdsresultsfile txt where e SDS RESULTS bat Is the name of the batch file you created e sdsfile sds Is the SDS plate document file that you have targeted for analysis e sdsresultsfile txt Is the name of the exported results file A 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Operating the SDS Software from a Command Line Enhancing the Itis possible to enhance the performance processing speed of the SDS software by Performance of increasing the memory partition allocated for the software To increase the memory the SDS Software made available to the SDS software insert the parameters shown in the example below into any command issued to the software jre bin java Dab home C AppliedBiosystems SDS2 2 service Dcom apldbio sds global approot Dcom apldbio sds global config config config properties Xms80m Xmx90m Dsource batchfile Ddefport 8 080 where e XmsS0m Is the minimum amount of memory that the operating system will allocate for the SDS software e Xmx90m Is the maximum amount of memory that the operating system will allocate fo
140. S software about 1 14 disconnecting 4 30 installing 7 55 launching 2 7 learning to use the software 2 10 re connecting 4 30 upgrading 7 55 Sealer installation 4 14 overview 4 14 Sealer see microfluidic card sealer Sealing guidelines 4 19 TaqMan Low Density Array 4 19 trimming fill reservoirs after sealing 4 21 selecting wells from the plate grid 2 24 Service Name A 22 setup table file about A 4 example files A 4 exporting A 17 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide importing into a plate document A 2 structure A 4 signal saturation 7 27 Slope readout from the Standard Curve Plot 6 13 SNP Manager Software 1 25 spectral calibration See pure dye runs Stage readout from the Real Time tab 4 27 Standard Curve Method 6 3 Standard Curve Plot about 6 13 exporting as a graphic file A 16 exporting data as a text file A 16 standards quantifying for absolute quantification B 6 selecting for absolute quantification B 6 selecting for relative quantification B 6 starting plate queue 4 43 run from the SDS software 4 25 State readout from the Real Time tab 4 27 Status readout from the Real Time tab 4 27 step adding to a method 3 19 Step readout from the Real Time tab 4 27 stopping plate queue 4 44 run from the SDS software 4 28 studies 1 28 study centric design 1 27 SYBR Green 1 Dye about D 3 System performance verifying RNase P card pr
141. Select File gt Save Results to Database 2 Inthe Description field of the Save Results to Database dialog box enter a brief description of the plate document up to 255 characters Note The software automatically populates the Session Name field with the name of the associated plate document 3 Optional In the Add to Study field click then select an existing study or create a new study by clicking nw and doing the following a Inthe Name field of the Configure the Create New Study dialog box enter a name for the study up to 128 characters The Creator field is not editable and displays your user name b In the Description field enter a brief description of the study up to 255 characters c Click d Click ox 6 52 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide After the Analysis 4 In the Save Results to Database dialog box click x 5 In the confirmation dialog box click x Saving the SDS 7900HT Document Although the SDS software saves the display settings and analysis settings to the plate document it does not save the results of the analysis You must analyze the plate document each time you want to review the analysis 1 Inthe SDS software select File gt Save As 2 In the Look in field of the Save As dialog box navigate to and select a directory for the software to receive the new file 3 In the File name field do one
142. Sk ES 5 10 Opening the Run Data lt 6 lt 4 icadecddda des teks eee ee edd ew eese a tee REs 5 11 Analyzing an Allelic Discrimination Run 0 0 0 0 cece eee 5 12 Calling and Scrutinizing Allelic Discrimination Data 44 5 13 PUY Te AUIS 65 5 4 44564 66059 240 ENEs EDEN NERIENE 1944 9094 8S E 5 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 5 5 Chapter 5 Analyzing End Point Data Overview Allelic Discrimination on the 7900HT Instrument Employing the 5 Nuclease Assay for Allelic Discrimination The Applied Biosystems 7900HT Fast Real Time PCR System supports allelic discrimination using TaqMan probes Allelic discrimination is the process by which two variants of a single nucleic acid sequence are detected in a prepared sample Allelic discrimination chemistry can be used for single nucleotide polymorphism SNP detection Allelic discrimination on the 7900HT instrument is made possible through the use of the fluorogenic 5 nuclease assay see page D 2 During the PCR the fluorogenic probes anneal specifically to complementary sequences between the forward and reverse primer sites on the template DNA Then during extension AmpliTaq Gold DNA polymerase cleaves the probes hybridized to the matching allele sequence s present in each sample The cleavage of each matched probe separates the reporter dye from the quencher dye which results in increased
143. System User Bulletin 2 Relative Quantification of Gene Expression PN 4303859 Note The normalized signal levels discussed above refer to the signals generated by the amplification of the target sequence in the unknown and calibrator samples which is normalized to the signal generated by the amplification of the endogenous control Note The SDS software does not support the Standard Curve Method of relative quantification Relative quantification on the 7900HT instrument is accomplished through the use of the polymerase chain reaction and the fluorogenic 5 nuclease assay After careful selection of an endogenous control probe and primer sets are designed for both the target and control sequences After the assays are verified unknown samples are loaded onto an optical plate containing master mix and 5 nuclease assays targeting a specific nucleic acid sequence The reaction plate is then run on a 7900HT instrument that is configured for real time analysis During the run the instrument records the emission resulting from the cleavage of TaqMan probes in the presence of the target sequence After the run the SDS software processes the raw fluorescence data to produce threshold cycle Cy values for each sample The SDS software computes relative quantities from the Cy values of the calibrator sample and the data from the unknown samples within the gene expression profile Note See Appendix D Theory of Operation for more inf
144. Time PCR System and SDS Enterprise Database User Guide Bibliography Bloch W 1991 A biochemical perspective of the polymerase chain reaction Biochemistry 30 2735 2747 Forster V Th 1948 Zwischenmolekulare Energie wanderung und Fluoreszenz Annals of Physics Leipzig 2 55 75 Higuchi R Dollinger G Walsh P S and Griffith R 1992 Simultaneous amplification and detection of specific DNA sequences BioTechnology 10 413 417 Higuchi R Fockler C Dollinger G and Watson R 1993 Kinetic PCR analysis Real time monitoring of DNA amplification reactions BioTechnology 11 1026 1030 Lakowicz J R 1983 Chapter 10 Energy Transfer In Principles of Fluorescent Spectroscopy Plenum Press N Y pp 303 339 Lee L G Connell C R and Bloch W 1993 Allelic discrimination by nick translation PCR with fluorogenic probes Nucl Acids Res 21 3761 3766 Livak K J Flood S J A Marmaro J and Mullah K B inventors Applied Biosystems Foster City CA assignee 2 Mar 1999 Hybridization assay using self quenching fluorescence probe United States patent 5 876 930 Livak K J Marmaro J and Todd J A 1995 Towards fully automated genome wide polymorphism screening letter Nat Genet 9 341 342 Martens H and Naes T 1989 In Multivariate Calibration John Wiley amp Sons Chichester General Papers de Kok J B Hendriks J C van Solinge W W Willems H L Mensink E J and on TaqMan
145. a Without a strategy for distributing and archiving SDS related files the 7900HT instrument can easily fill the hard drive of the computer within just a few weeks of operation See Managing Sequence Detection System Data on page 1 17 for a discussion of management strategies Archiving To conserve space on the computer hard drive SDS files can be archived using a data SDS Files compression utility The compression utility archives files by encoding them in a compressed form thereby reducing the size of a file SDS files can be compressed and decompressed many times Several commercially available compression utilities are available PK ZIP and arc are archive formats common to the Microsoft Windows operating system Defragmenting Applied Biosystems recommends defragmenting the hard drive of the computer the Hard Drive attached to the instrument at least once every week or before fragmentation reaches 10 As the Applied Biosystems 7900HT Fast Real Time PCR System is used and files are deleted and created the free space on the computer hard drive eventually is split into increasingly smaller blocks called clusters Consequently as the SDS software creates new files and extends old ones the computer cannot store each file in a single block Instead the system will fragment the files by scattering their component pieces across different sectors of the hard drive The fragmentation of SDS files decreases the performance of
146. a collected by the 7900HT instrument The overlapping spectra from the individual dye components in a well generate the composite spectrum that represents a raw data fluorescent reading A technique that uses multiple fluorogenic dye labeled TaqMan probes to detect the simultaneous amplification of two or more target nucleic acids within a DNA sample A control reaction with all PCR components included except DNA template After a detector is applied to a well each detector must be assigned a task that describes its purpose One of the tasks is NTC and is applied to all detectors of negative control wells containing PCR reagents but lacking samples See R One of the structural components or building blocks of DNA and RNA A nucleotide consists of a base one of four chemicals adenine thymine guanine and cytosine plus a molecule of sugar and one of phosphoric acid An unusually extreme value for a variable given the statistical model in use In the SDS software automatic outlier removal in RQ Studies is conducted to assist in the elimination of data contamination occurring when a process or phenomenon other than initial sample quantity affects the measured Cy A reagent mix containing all components necessary for PCR except template DNA and primers probes For example the TaqMan 2X Universal PCR Master Mix contains AmpliTaq Gold DNA polymerase AmpErase UNG that protects against carry over contamination Passive Reference I fo
147. a cycle range of high precision during which amplification is characterized by a high and constant efficiency It occurs between the first detectable rise in fluorescence and before the beginning of the linear phase When plotted on a log scale of DNA vs cycle number the curve generated by the geometric phase should approximate a straight line with a slope The 7900HT instrument typically delivers sufficient sensitivity to detect at least 3 cycles in the geometric phase assuming reasonably optimized PCR conditions D 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Real Time Data Analysis Phase 2 Linear The linear phase is characterized by a leveling effect where the slope of the amplification curve decreases steadily At this point one or more components have fallen below a critical concentration and the amplification efficiency has begun to decrease This phase is termed linear because amplification approximates an arithmetic progression rather than a geometric increase Because the amplification efficiency is continually decreasing during the Linear phase it exhibits low precision Phase 3 Plateau Finally the amplification curve achieves the plateau phase at which time the PCR stops and the R signal remains relatively constant Determining At any given cycle inside the geometric phase of PCR the amount of product is Initial Template proportional to the initial number of template copi
148. aa e Bak ati Ded a Bok dle Pee ed eee a a ani A 4 NaS Veron narte ieee sraa e Seah os ee see ot ow Oe asd ee dee A 5 Plate GHarACtCNSNCS eeen saan ete eae whl a See ele See See ake eae as A 5 ZPE SILC e s sos hE a aR A EAS A ONO Ee ERE ee See ae A 5 eM Ae sce araa Bion a eae ats ne deep atte wey Nerd ene a a E tava eaten lace A 5 Detector Definitions 0 0c eee eas A 6 4 Number of Detectors 0 00 eee A 6 5 Detectors Lst Header errs Sts ee ae Be a la a a es a eae 2 A 6 6 Detectors EIST 22ch 3620042 wt erie A eee wee Phu aes Lee A 7 Marker IDSTIAIIOINS s cucs ia ides ser di Sen l a aE AE an me eon a A 8 7 Number of Markers Allelic Discrimination Only 00200 e eee A 8 8 Markers List Header Allelic Discrimination Only 000000e A 8 9 Markers List Allelic Discrimination Only 00 0c eee eee eee A 9 VWell DeTeClOr IMNOMMAUON sucess lute heivew eds ones Poewe Dees Loew iS SeeA A 10 10 Well Detector List Header 0 0 0 0 ccc es A 10 11 Well Detector Definition List 0 0 0 0 ccc eee A 10 Well Marker Information 0 0000 ce eee eee ees A 12 12 Well Marker List Header Allelic Discrimination Only A 12 13 Well Marker Definition List Allelic Discrimination Only A 12 Example Setup lable Files 22 12 4dn teiutaeeeeid eed be ae ee E eee A 14 EXDOring Grapes 4 4 02 02440 tAn 67 ntewaee aoe eens 45645 0 0 Gamat eucra
149. ach pad thoroughly with Isopropanol until the residue has been resolved If the pads appear rough or the adhesive cannot be removed replace the pads as described below 1 Using the Philltps head screwdriver remove the two small Phillips head screws from the fingers on each side of the gripper then remove the fingers Note Move the Plate Handler arm into any position where it is easy to access the screws 2 Using a small flat blade screwdriver pry the worn finger pads off the fingers Note The manufacturer recommends replacing all finger pads at the same time 3 Clean any residual adhesive off the fingers using isopropanol 4 Remove a replacement finger pad from the paper backing and place the finger pad on the appropriate finger position 5 Repeat for the remaining finger pads 6 Install the fingers with the fingers pointing down and the finger pads toward the center of the gripper 7 Insert the screws into the fingers and tighten Note The screws do not automatically align the grippers Make sure that the finger pads are making good contact with the plate when the arm grips a plate 7 52 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 7 3 Maintaining the Computer and Software Section 7 3 Maintaining the Computer and Software In This Section This section contains the following information General Computer Maintenance 0 00 ccc ees 7 54 Maintain
150. action mix master mix RNase P primers and FAM dye labeled probe and template to detect and quantitate genomic copies of the human RNase P gene 1 x 96 Well Plate 4323306 TaqMan RNase P 384 Well Instrument Verification Plate Includes one ABI PRISM 384 Well Optical Reaction Plate Each well contains preloaded reaction mix master mix RNase P primers and FAM dye labeled probe and template to detect and quantitate genomic copies of the human RNase P gene 1x 384 Well Plate Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide C 5 Appendix C Kits Reagents and Consumables Table C 3 Consumables for Instrument Maintenance and Verification Part N mbEr Description Quantity 4351979 TaqMan RNase P Fast 96 Well Instrument Verification Plate 1 x 96 Well Includes one Optical 96 Well Fast Thermal Cycling Plate PIRRE Each well contains preloaded reaction mix master mix RNase P primers and FAM dye labeled probe and template to detect and quantitate genomic copies of the human RNase P gene TaqMan Low Density 7900HT Installation Array TGF B card TaqMan Pre Developed Assays and Reagents For the latest information on TaqMan PDARs covering gene expression quantification and allelic discrimination visit the TaqMan PDAR list on the Applied Biosystems web site at e www appliedbiosystems com pdarlist Custom Oligonucle
151. action plate in the input stack 1 of the Plate Handler Power on the 7900HT instrument the Plate Handler and the computer Select g amp stat gt amp Programs gt amp Zymark Twister Plate Handler gt Twister The Zymark Twister Software starts Click Manual Control In the Manual Control dialog box click stack 4 Rotary Positioning Click 4 Rotary Adjustment Je The Plate Handler arm moves over the input stack Click amp Find Plate If the adjustment was successful the Plate Handler arm will lower upon the plate until the plate detector switch engages confirming the presence of the plate If Plate Handler arm emits a grinding sound adjust the plate sensor switch a In the Zymark Twister Software click t Vertical Home to raise the Plate Handler arm b Turn the thumbscrew in the Down direction 10 steps c Repeat step 6 until the Plate Handler arm successfully detects the plate p Click Close Gripper then click Vertical Home If the adjustment was successful the Plate Handler arm will grasp the plate and remove it from the plate stack If Plate Handler arm stops before the gripper fingers are able to contact the plate and fails to grasp or pick up the plate adjust the plate sensor switch a Turn the thumbscrew in the Up direction 10 steps b Grasp the plate with one hand and from the Zymark Twister Software click pen Gripper to release the plate c Replace the reac
152. adding plate documents to the queue the plate documents automatically become available for use with the Automation Accessory IMPORTANT After you have added a plate document to the plate queue the software locks the file preventing any changes from being made to it until you run or remove the plate document from the queue The Applied Biosystems 7900HT Fast Real Time PCR System has three options for adding plate documents to the plate queue Review the options listed below then choose the method that best suits your needs e Add a plate document to the plate queue from the SDS software page 4 35 e Using the Template Batch utility create batches of plate documents from a plate document template and add them to the plate queue page 4 36 e Add or remove individual or multiple plate documents to the plate queue using the Automation Controller Software page 4 39 1 Add plate documents to the plate queue Add a plate document to the plate queue using the SDS Software see page 4 35 Create and add plate documents to the plate queue using the Template Batch Utility see page 4 36 2 Start and configure the Automation Controller Software and add or remove plate documents from the plate queue as needed See page 4 39 3 Load prepared optical plates and Low Density Arrays onto the stacks of the Zymark Twister Microplate Handler see page 4 41 4 Run the instrument see page 4
153. adioactive or biohazardous materials may require special handling and disposal limitations may apply Electrical Safety Fuses Power XX j DANEJ ELECTRICAL SHOCK HAZARD Severe electrical shock can result from operating the Applied Biosystems 7900HT Fast Real Time PCR System without its instrument panels in place Do not remove instrument panels High voltage contacts are exposed when instrument panels are removed from the instrument DNIe das ELECTRICAL SHOCK HAZARD Improper fuses or high voltage supply can damage the instrument wiring system and cause a fire Before powering on the instrument verify that the fuses are properly installed and that the instrument voltage matches the power supply in your laboratory VERONI FIRE HAZARD For continued protection against the risk of ire replace fuses only with fuses of the type and rating specified for the instrument ag DANa ELECTRICAL HAZARD Grounding circuit continuity is vital for the safe operation of equipment Never operate equipment with the grounding conductor disconnected j DANa ELECTRICAL HAZARD Use properly configured and pproved line cords for the voltage supply in your facility fly j DANa ELECTRICAL HAZARD Plug the system into a properly grounded receptacle with adequate current capacity Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Physical Hazard Safety Overvoltage The Applied
154. alized reporter dye signal for example the ratio of FAM to VIC dye The result of the analysis typically yields three major clusters corresponding to the three genotypic constituents Allele X homozygous Allele Y homozygous and heterozygous After clustering the data points the software applies a series of probability tests to further refine the clustered data and to identify outliers For every sample the software calculates a final quality value that represents a measure of the likelihood that the sample belongs to each cluster The algorithm then applies the quality value for the marker set in the Analysis Settings dialog box as a threshold for calling the associated sample data Sample cluster associations that generate quality values greater than the defined threshold are automatically assigned the call of the associated cluster Note Any SNPs which depart substantially from expected Hardy Weinberg frequencies should be reviewed About the 2 Cluster Calling Feature If only two clusters are present in a study the AutoCalling algorithm uses the expected genotype frequencies of the clusters to assign their genotypes If a study contains two large clusters and a single sample between the two clusters the software considers the intermediate sample as an outlier rather than a cluster and treats the data as a two cluster study Note The Maximum Likelihood Algorithm is designed to use No Template Controls and autocalling is
155. an Sha edk a ceee ek Oe ae eT ROE GAS E Saa 8 6 TT M oes pees eh a ce owes bee eee eee Bee ee eae ome 8 6 Pl Pe ot eae E bee keke Gad dy bee Mee nwt ea eee ne 8 6 Splashing PCR Reagents o coeds deeseedsageeads ehbeuaew des TEEPEE 8 6 Eoee E SE EE E E E E E E E E E E eae eas 8 7 Writing on the Reaction Plates 0 0 cc eens 8 7 Fluorescent Contamination on the Plates 20 0 0 8 7 Bes a eee ee ee ee eee eee eee 8 7 Contaminated Sample Block 64 44 0042 0400084 006 60 5e 08s 4Ob bed eae SS 8 7 Improper or Damaged Plastics 26 c4546 oy dbieveaet hag eed beedecdae 8 8 LOW Copy Emplis cae ec rines kariad deeeouredes barked dee e ohne ee 8 8 Use of Non Applied Biosystems PCR Reagents 0000008 8 8 The key to high precision quantitative PCR is accurate detection of the geometric phase The Applied Biosystems 7900HT Fast Real Time PCR System typically delivers sufficient sensitivity so that at least three cycles of the geometric phase are visible assuming reasonably optimized PCR conditions The SDS software calculates a fixed signal intensity called a threshold that each signal generated from PCR amplification must reach before it is recognized by the software as actual amplification The calculated threshold is an approximation and should be examined and modified as needed Modifying the Threshold In a real time document of the SDS software the threshold can be modified via the Amplification plot view follo
156. analyses of SNP genotyping data stored on the database The software can make comparative analyses of up to 5 000 plates or approximately 1 9 million 5 nuclease reactions run on an Applied Biosystems 7900HT Fast Real Time PCR System See the SNP Manager Software User Guide PN 4351671 for more information The RQ Manager Software is a stand alone application used to perform medium to large scale analyses of relative quantification gene expression data stored on an SDS Enterprise Database The software can make comparative analyses of over 97 detectors 96 target genes and 1 endogenous control across 200 plates or approximately 153 600 multiplexed reactions run on an Applied Biosystems 7900HT Fast Real Time PCR System See the RO Manager Software User Guide PN 4351670 for more information 1 25 Chapter 1 Product Overview Database Management Utilities Functional Summary i keJ Plate Documents SDS Document Creator re keJ Plate Documents SDS Document Command Line Tools SDS Database Archiver 1 26 gt The SDS Enterprise Database includes a suite of software applications that administer the use and content of the database Figure 1 17 summarizes the basic function of each tool and the type of data it handles SDS SDS Ba ate Document Template Documeni Loader Loader Templates SDS User Account Plate Manager Documents SDS Enterprise Database SDS
157. analysis the software mathematically transforms the raw data to establish a comparative relationship between the spectra changes in the passive reference dye and those of the reporter dye Based on that comparison the software calculates a cycle threshold Cy for each reaction endogenous control and unknown Note See Appendix D Theory of Operation for a detailed description of the SDS software mathematical transformation of real time run data l Click or select Analysis gt Analyze The SDS software analyzes the run data and displays the results in the Results tab Choose from the following e Ifyou choose to set the baseline and threshold values manually set them for each detector on the plate as explained on page 6 46 e Ifusing the AutoCy algorithm to set the baseline and threshold values for the detectors on the plate verify that the software has called the settings correctly for each detector by following the procedure for manually setting the baseline and threshold on page 6 46 without adjusting the settings If you choose to remove outliers manually visualize and eliminate outliers from the analyzed run data as explained on page 6 46 6 30 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Viewing Results Viewing Results Displaying the The Gene Expression plot displays the data of the selected target detector s in the Analyzed Data Detectors list The SDS sof
158. and SDS Enterprise Database User Guide About the SDS Enterprise Database Software Suite About the SDS Enterprise Database Software Suite Database Management Software The SDS Enterprise Database provides software utilities to help integrate the database into laboratory workflows as describes in Database Modules for Large Scale Analysis Database Management Utilities Database Modules for Large Scale Analysis About the Analysis Modules About the SNP Manager Software About the RQ Manager Software Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Applied Biosystems offers two analysis modules the SNP Manager Software and RQ Manager Software for conducting medium to large scale analysis of SNP genotyping and gene expression on the SDS Enterprise Database Figure 1 16 Allelic Discrimination 17 4 86a SNP m P Manager a s Applied Biosystems SNP Manager Software 8 1 3 at 3 a AR 35 SNP Genotyping Study Amplification Plot Gene Expressi ion l Studies Run Data SDS Enterprise Database Analysis Sessions sada RO Manager Applied Biosystems RQ Manager Software Relative Quantification Study Figure 1 16 SDS Enterprise Database Applications for Large Scale Analysis The SNP Manager Software is a stand alone application used to perform medium to large scale
159. and reverse primers and FAM and VIC dye labeled TaqMan probes Figure 5 5 illustrates the arrangement of the assays unknown samples and no template control NTC wells on the plate O So O Senne EEE GR2107 PD PDAR CYP 2C9 2 PDAR CYP 2C19 2 Figure 5 5 Configuration of the Samples and Detectors on the Example Plate Note The probes used in the example experiment were designed using the Primer Express Primer Design Software and by following the guidelines explained in Assay Development Guidelines on page B 2 IMPORTANT The SDS software does not require that absolute quantification plates contain positive controls Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 5 9 Chapter 5 Analyzing End Point Data Analysis Checklist Where You Are in the Procedure 1 Analyze the run data See page 5 11 2 View the results of the allelic discrimination run see page 5 13 3 Call allele types for each marker see page 5 14 4 Scrutinize the allele calls See page 5 16 Y 5 Choose from the following post analysis options see page 5 18 Reanalyze the run data Adjust the display settings for the plate document Print elements of the plate document Export the plate document results table or plots 5 10 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Databa
160. anes Views elements of the plate document When clicked a sizing button hides the element to and Plots which it points 1 Click down arrow sizing button in the divider between the plate grid and the table pane to maximize the plate grid vertically The software maximizes the plate grid by minimizing the table pane Fie Et Yew Tod out Ansty Ando TA Haig Uh 2 2s SR rr SDT a aa Bm E Practice Abseleb arstina SE jaja spas e rja epi az ae ts faeieel ae f Stor rete SRS S BOSSE E E an a BBE RE RRR ERR BBB BBD D sere re i rete a aa f tite Getemor Reno Tiii nay scolar CER ERR RRR EERE EEE a pre Ea a EB REE EEE RE RBERE RES ES os HRB ER EERE EERE BEBE Sizing button TTT TTLVTILILLIELLEILLEL A HE BRE ERE EERE 1 RHE Hna Gham Gham Gham EHowe RHE RHE Y RHaaF Starcard ST 2 Click and drag the grey divider at the bottom of the plate document to restore the table pane to its original size using the action described in Exercise 1 i SOS 2 Fi Edt View Tous Point Anyar Aniw i h oe i Fractie Absebebe Desain aii Maiy it a TE oM E a e a Eaa A ll eee eee LSe am oo a O X l X l l l l aaa Z eee a _ e a a eer e e_me a m e ar a rt tt 4 Ae te eS ee SS SS SS eS SS eS l e e Te ee eS SS E a E jp PEE EERE REE ERRERREEE 3 Using the sizing buttons maximize minimize other elements of the plat
161. ark Twister Software click position 4 b Click FindPlate C Click Close Gripper Click position 2 Click here The Plate Handler arm moves over the instrument tray Use Vertical Positioning to lower the Plate Handler arm until it is approximately 1 cm above the instrument tray Using the Rotary Adjustment arrows center the gripper and plate along the Y axis of the instrument tray Center the plate o Center the gripper and plate along the X axis of the instrument tray by sliding the Plate Handler and base towards or away from the 7900HT instrument Center the plate 6 Again using the software to move the Plate Handler arm center the gripper and plate along the Y axis of the instrument tray as explained in step 4 7 44 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 10 11 Aligning the Plate Handler Using the Vertical Positioning commands carefully lower the Plate Handler arm onto the instrument tray and confirm that the plate rests squarely inside it Tighten the three black thumbscrews on the platform connecting the 7900HT instrument and the Plate Handler Release the plate from the Plate Handler arm d Click Open Gripper b Click
162. arted Saving the Plate Document to the SDS Enterprise Database 1 Click AJ or select File gt Save Document to Database 2 In the Document not properly set up warning click Document not properly set up E x AN This plate does not contain any detector information It cannot be analyzed until detectors are defined and added to wells 3 Inthe Save Document to database dialog box leave the Comment field blank or enter a brief comment describing the file Note The Comment field can contain up to a 256 character description of the file Save Document to Database l a E x Unique Mame or Barcode Practice Comment Ps is a practice document 4 Click to save the file 5 In the Document not properly set up warning click Document not properly set up E x IN This plate does not contain any detector information It cannot be analyzed until detectors are defined and added to wells 7 Select File gt Close 8 If prompted to save the plate document click _ te The SDS software closes the file without saving it 9 Open a plate document file as explained on page 2 19 2 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Basic Software Skills Tutorial Exercise 5 In this exercise you will be opening two documents that you will use in the following Opening a Plate exercises the plate document from Exercise 3 and a plate document templ
163. ase User Guide 4 23 Chapter 4 Operating the Instrument Saving the Plate Document User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to save a plate document to the database Saving the Plate Before the plate document can be run you must save it to the SDS Enterprise Document for Database or as an SDS 7900HT Document sds Single Plate Operation To save as an SDS 7900HT Document l 2 5 In the SDS software select File gt Save As In the Look in field of the Save As dialog box navigate to and select a directory for the software to receive the new file In the File name field either e Enter a file name for the plate document file or e Enter or scan the bar code number for the plate into the field Note The SDS software does not require that the file name match the bar code of the corresponding plate Click se The software saves the plate document to the specified directory Run the plate document and associated plate as explained on page 4 25 To save to the SDS Enterprise Database l 2 3 Select File gt Save Document to Database Configure the Save Document to Database dialog box a In the Comment field enter a brief description of the plate document up to 255 characters If saving a plate document file to the database you will loose the file name unless you enter it into the Comments field b Cl
164. assword Figure 7 2 Electronic Signature Verification Dialog Box Options Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Recommended Maintenance Schedule Recommended Maintenance Schedule Maintenance The following table includes a list of tasks that should be performed on the Schedule Applied Biosystems 7900HT Fast Real Time PCR System regularly Table 7 1 Maintenance Schedule for the 7900HT Instrument Interval Task See Page SuM T WThF S Archive SDS Files 7 54 Week 7 Days SuM TWThF S Perform a Background Run 7 16 Check and if necessary replace Gripper Finger 7 52 Pads Month 30 Days Defragment the Computer Hard Drive 7 54 July Perform a Pure Dye Run 7 20 Check Applied Biosystems Web Site for Software 7 55 Updates Semi Annually 6 Months Perform a background run before each Pure Dye Run Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 3 Chapter 7 Maintaining the Instrument 7 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 7 1 Maintaining the 7900HT Instrument Section 7 1 Maintaining the 7900HT Instrument In This Section Replacing the Sample Block 00 00 00 en 7 6 C Wai the Plate Ae ook eo dG Oh d
165. ast 96 Well Block Remove the sample block from the 7900HT instrument as explained in Removing the Sample Block on page 7 7 Use the following figure as a guide to locate the suspected contaminated wells on the sample block A rnin 7 Circuitry and connections to the instrument Do Not Touch Well A 1 GR2028 Pipette the appropriate volume of 10 bleach solution into each suspected contaminated well of the sample block module e For a Standard 384 Well Block pipette 40 uL bleach solution to each well e For a Standard 96 Well Block pipette 150 uL bleach solution to each well e For a Fast 96 Well Block pipette 55 uL bleach solution to each well Allow the sample block to sit for 3 to 5 min Using a pipet remove the bleach solution from the wells of the sample block Rinse pipette and remove each contaminated well with three treatments of deionized water at the appropriate volume for the sample block e For a Standard 384 Well Block rinse affected wells with 40 uL deionized water e For a Standard 96 Well Block rinse affected wells with 150 uL deionized water e Fora Fast 96 Well Block rinse affected wells with 55 uL deionized water Note Absolute isopropanol can be substituted for water in the third treatment Remove any remaining isopropanol or water from the wells of the sample block module Replace the sample block as explained in Re
166. ast Real Time PCR System and SDS Enterprise Database User Guide 2 11 Chapter 2 Getting Started Lesson 1 Using Plate Documents About SDS Plate Every plate run on the Applied Biosystems 7900HT Fast Real Time PCR System Documents _ requires the creation of a plate document using the SDS software A plate document is a virtual representation of a consumable used to contain samples and reagents during a sequence detection run The software uses the plate document to coordinate the operation of the instrument thermal cycling and data collection to organize and store the data gathered during the PCR and to analyze the run data Table 2 3 Plate Document Types of the SDS Software File Ext Icon Description SDS 7900HT sds Plate Documents are the primary file you will use Document LR They are generated for every kind of experiment and are generally used to run plates SDS 7900HT sdt Although optional plate document templates are Template F useful as timesaving devices for experiments where Document samples are run on plates with identical assay configurations SDS 7900HT sdm pe Multiple plate documents are used to analyze data Multiple Plate LR from relative quantification experiments The use of Document multiple plate documents is discussed in detail in Chapter 6 Analyzing Real Time Data The exercises in this lesson will familiarize you with the use of plate documents 2 12 Applied
167. ata from a set of dye standards during a 2 min hold at 60 C The software stores the spectral information for the pure dye standards in a calibration file located in the SDS directory After the run the software extracts each component dye spectrum from the collected data in the pure spectra run file IMPORTANT Because the age and use of instrument components can affect pure spectra readings Applied Biosystems recommends updating the pure spectra data files once or twice annually depending on instrument use The Applied Biosystems 7900HT Fast Real Time PCR System monitors fluorescent signals generated by several dyes inside the preloaded pure dye plate For the standard 384 well standard 96 well or Fast 96 well pure dye plates the dyes are FAM NED ROX SYBR Green TAMRA TET and VIC For the 7900HT System TaqMan Low Density Array Upgrade the 7900HT instrument monitors fluorescent signals generated by three dyes FAM VIC and ROX Your installation kit contains these dyes and you introduce them into three separate microfluidic pure dye cards to perform pure dye runs 0 00 O Dye Peak nm MAVA y O TS 3 TET 540 4 VIC 590 JOE 550 l 5 NED 570 6 TAMRA 580 7 ROX 610 Figure 7 4 Standard Pure Dye Spectra of the 7900HT Instrument Note The 7900HT instrument supports the detection of custom pure dyes dyes other than those provi
168. atabase Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 19 Chapter 1 Product Overview 1 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 1 3 SDS Enterprise Database Section 1 3 SDS Enterprise Database In This Section About the SDS Enterprise Database Feature 00 00 0000 ccc eee 1 22 About the SDS Enterprise Database Software Suite 000 1 25 Database Design and Information Management 000 00 ae 1 27 Supporting API Documentation nnana aeaaaee aeaaea 1 28 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 21 Chapter 1 Product Overview About the SDS Enterprise Database Feature About the Database Access Control Network Architecture The SDS software can interface directly with an Applied Biosystems SDS Enterprise Database as part of a laboratory information management system LIMS The SDS Enterprise Database is a relational Oracle based repository designed specifically to store SDS data produced by an Applied Biosystems 7900HT Fast Real Time PCR System In addition to serving as a common repository for SDS data the database provides user authentication robust and scalable data management and flexible archive capabilities The database also provides the infrastructure and tools necessary to integrate a 7900HT instrument into a laboratory
169. ate The Document purpose of this lesson is to familiarize you with the tasks of retrieving plate document that have been saved to the computer hard drive or the SDS Enterprise Database Choose from the procedures below e Ifusing a database save the plate document to the database as described on page 2 18 e Ifyou are not using a database save the plate document to your computer hard drive as described below Opening the SDS Plate Document File 1 Click or select File gt Open 2 In the Look In field of the Open dialog box navigate to Applied Biosystems SDS Documents 3 In the Look In field select the Practice sds file Look in SD5 Documents E j g Reece cae a A A AA AAA AEA E Sel ect File name Practice sds Open Files of type aer Prism 505 Document sds Cancel 4 Click open The software opens the Practice sds file 5 Select File gt Close Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 19 Chapter 2 Getting Started 2 20 Opening the Plate Document from the SDS Enterprise Database 1 Click or select File gt Open Document from Database 2 Configure the Find Plates Matching These Criteria table of the Plate Query dialog box a Click the first cell in the Variable column and select Barcode b Click the first cell in the Conditions column and select Contains c Click the first cell in the Valuel column and enter
170. ates or loading Practices TaqMan Low Density Arrays for use on the 7900HT instrument e Wear a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves when preparing samples for PCR amplification e Change gloves whenever you suspect they are contaminated e Maintain separate areas dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products e Never bring amplified PCR products into the PCR setup area e Open and close all sample tubes carefully Try not to splash or spray PCR samples e Keep reactions and components capped as much as possible e Use positive displacement pipets or aerosol resistant pipet tips e Clean lab benches and equipment with 10 bleach solution Compatible Consumables Consumables for Table 4 1 Compatible Consumables Use with the 7900HT Compatible Seals Instrument Consumable Type Use these Not these ABI PRISM e ABI PRISM Optical Adhesive Covers 384 Well Septa 384 Well Optical quantity 100 Reaction Plates e Optical Adhesive Covers quantity 25 4 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Table 4 1 Compatible Consumables Compatible Consumables Consumable Type Compatible Seals Plates ABI PRISM Snap On Compression Pad reusable 96 Well Plate Seals ABI PRis
171. ation The results of the experiment can be visualized in the Standard Curve graph of the Results tab The graph consists of a scatterplot of standard and unknown samples graphed on a linear scale plot of Threshold Cycle C versus quantity value Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 7 Chapter 6 Analyzing Real Time Data Before You Begin Using the SDS For specific instructions on any procedure described within this section refer to the Software Sequence Detection Systems Software Online Help To get help at any time during the Online Help procedure click 17 located within the dialog box or window in which you are working Examples in This The illustrations and screenshots that appear within this chapter were created for a Chapter TaqMan RNase P 384 Well Instrument Verification Plate PN 4323306 an experiment run during the installation of the 7900HT instrument to verify its performance The sealed plate is preloaded with the reagents necessary for the detection and quantification of genomic copies of the human RNase P gene a single copy gene encoding the RNA moiety of the RNase P enzyme Each well contains pre loaded reaction mix TagMan 2X Universal PCR Master Mix RNase P primers and FAM dye labeled probe and template Figure 6 4 illustrates the arrangement of standards and samples on the RNase P Instrument Verification Plate As shown below the RNase P Instrument Veri
172. ation Plate can be ignored to meet specifications 7 34 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 7 2 Maintaining the Plate Handler Section 7 2 Maintaining the Plate Handler In This Section Automation Accessory Components and Stack Positions 7 36 Adjusting the Sensitivity of the Plate Sensor Switch 0004 7 37 Algning the Plate Handler iy hee ood eww eo es aisats i sadaa KS OS 7 41 Aligning the Fixed Position Bar Code Reader 0 0 00 0c eee 7 49 Cleaning and Replacing Gripper Finger Pads 0 0 00 e ee euee 7 52 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 35 Chapter 7 Maintaining the Instrument Automation Accessory Components and Stack Positions Automation Refer to Figure 7 5 for the components discussed in this section Accessory Components cross sectional view of the gripper Plate sensor Gripper Adjustment switch knob Plate stack Expansion stacks Zymark Twister Microplate Handler Fixed position bar code reader Figure 7 5 Components of the Automation Accessory Plate Stack The Zymark Twister Microplate Handler alignment is performed using the Zymark Positions Twister Software The software refers to the positions of th
173. ation dialog box with the current configuration settings 4 Configure the software for the alignment a In the bottom of the Edit Configuration dialog box locate and select the Op Modes tab Op Modes tab Note You may need to use the arrows located in the bottom of the dialog box to locate the Op Modes tab b In the Operating modes selection group of the Edit Configuration dialog box click the arrow to the right of the Mode heading and select Test from the drop down list EA LD11000 Edit Configuration ADIGE Jaz Operating modes selection Serial on line x Device Configuration Online erial on line Automatic i est ieee Mode drop down list is eee eee ae eee c In the Device Control dialog box click RAM to toggle to EEPROM mode r Device Control Eel x Device LD11000 Port COM4 9600 N81 Status W Tx RX Commands Iili Verifier d In the Device Control dialog box click Send e In the Confirm dialog box click YES to save to EEPROM The fixed position bar code reader begins a continuous repeating scan of the bar code The software updates the Terminal dialog box every 0 5 sec indicating the percentage of accurate reads completed during the 0 5 sec interval RAM button 7 50 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 10 Aligning the Fixed Position Bar Code Reader Loosen the black positional adjustment knob on the f
174. ation without the database see page 4 34 3 Analyze the run data Allelic Discrimination Analysis See page 5 5 Absolute Quantification Analysis See page 6 5 Relative Quantification Analysis See page 6 15 Dissociation Curve Analysis See page 6 37 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 3 Chapter 4 Operating the Instrument 4 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 4 1 In This Section Section 4 1 Consumable Preparation Consumable Preparation Preventing OIA ik oo 5 5 4 4 9496994 OE bd ORE ES 4 6 Compatible CASS 4 6c 5 6 oboe 84 ee ee oe oe AERE 4 6 Preparing Optical Plates for USG g4 4 e5 bd RSE EOSS 6 O54 Fh OO eee ES 4 8 Preparing TaqMan Low Density Arrays for Use 20 0 0000 eee ee 4 10 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 5 Chapter 4 Operating the Instrument Preventing Contamination Contamination PCR using the 5 nuclease assay requires special laboratory practices to avoid false and the positive amplifications Kwok and Higuchi 1989 The assay s logarithmic 5 Nuclease amplifications can potentially amplify a single DNA molecule Saiki et al 1985 Assay Mullis and Faloona 1987 General PCR Observe the following guidelines when assembling optical pl
175. au When using the instrument in its Plate Read mode end point analysis the fluorescence signal is read at a single point in time after the completion of PCR rather than at intervals during the course of PCR A run describes a collection of fluorescence data carried out by the 7900 instrument during and or after cycling of samples in an optical plate or TaqMan Low Density Array See Analysis Session A single base pair of DNA in the genome that differs between individuals A PCR reaction with a single fluorescent detector per well See single nucleotide polymorphism A portion of the complete range of the wave length measured by the 7900HT instrument approximately 500 660 nm Spectral calibration occurs from pure dye results obtained from a pure dye run The software stores the spectral information for the pure dye standards within a calibration file located in the SDS directory After a run the software extracts each component dye from the collected data in the pure spectra run file Because the age and use of the instrument components can affect pure spectra readings Applied Biosystems recommends updating the pure spectra run file once or twice annually depending on instrument use The fluorescent contribution of one dye signal to another caused by the overlap of their spectra The physical container of a 7900HT instrument into which well plates can be stacked There are 5 stacks in all Four of the five consecutively numbered
176. ay settings you must save the plate document to the database for the changes to persist select File gt Save Plate Document to Database e The SDS Enterprise Database allows two or more users to open and modify data simultaneously If another user has opened analyzed and saved the same data that you have just analyzed the software overwrites their analysis when you save yours to the database e Analysis session results are directly linked to the configuration of the plate document used to create the session If after you have saved your analysis session to the database you modify the linked plate document by omitting wells altering sample names etc your analysis session may be orphaned 1 e have no relationship to its attached plate document Consequently the results of your analysis session may change if you reanalyze it because you have changed the content of the associated plate document containing the raw data Applied Biosystems recommends the following guidelines for using analysis sessions Save the plate document as a new document any time you make changes to the plate setup omit wells alter sample names modify detectors or markers etc Maintain only one saved analysis session per document Understand that only results sessions saved after the latest changes to a plate setup are guaranteed to correspond to the linked plate document To save the save the analysis session to the database 1
177. b Auto Increment tab Ramp Rate tab Data Collection tab e Real Time tab e Plate Read tab Raw Data Plot Displays the raw fluorescence collected from the sequence detection run The Raw Data tab is visible only in plate documents containing run data To view the tab open a plate document containing run data click Bi then select the Raw Data Plot tab Raw Data Plot Calibration Data Displays the Background and Pure Spectra calibration data used for the signal normalization and multicomponenting analysis of the run data The Calibration Data tab is visible only in plate documents containing run data To view the tab open a plate document containing run data click Bi then select the Calibration Data tab Calibration Data e Background Plot e Pure Dyes Plot Results Dissociation Curve Displays analyzed run data The Analysis tab is visible only in plate documents containing analyzed run data Displays analyzed dissociation curve data from a programmed ramp The tab is visible only in plate documents containing analyzed data from a real time run with a programmed ramp Allelic Discrimination Data e Allelic Discrimination Plot Absolute Quantification e Amplification Plot e Standard Curve Plot Relative Quantification e Amplification Plot Dissociation Plot 2 30 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide
178. below for use in Japan IEC 320 Japan x 1 In Bag Marked JAPAN ONLY North America 250V 15A MAX N oy i 0 N Europe and all other locations 250V 10A MAX Figure 1 10 Universal Power Strip IEC 320 U K lreland x 1 To computer monitor hand held bar code reader and the Plate Handler IEC 320 Australia New Zealand x 1 Note The order is not critical To install the Universal Voltage Accessory Kit All Countries Except Japan 1 Connect one universal jumper to each accessory see below for use in Japan 2 Connect the other end of the power jumpers to the power strip outputs 3 Choose the correct country specific power input cord for the geographical region and connect it to the power strip input 4 Power off all instrument accessories monitor computer and Plate Handler 5 Connect the power input cord to the AC outlet 6 Power on accessories 7 Discard unused cord sets Usage Guidelines for Japan 1 Use only the cord sets supplied in the bag marked JAPAN ONLY 2 Discard all other unused cord sets Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 11 Chapter 1 Product Overview 1 12 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 1 2 Getting to Know the Software Section 1 2 Getting to Know the Software In This Section Software Components 0 0 0 0000 ccc ccc cece eee eee 1 14 SDS S
179. c discrimination absolute quantification or relative quantification Method setting that increases or decreases the value for any PCR set point parameter time or temperature by a fixed amount every cycle Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide background baseline calibrator cDNA complementary DNA clipped data command line consumable container type cycle set point delta C delta delta C delta R Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide An inherent spectral component of the signal generated by a fluorescence assay that is attributed to the Applied Biosystems 7900HT Fast Real Time PCR System Uncorrected background signal can interfere with the sensitivity of the SDS software and its ability to determine Cys For this reason background signal is measured during calibration and is removed mathematically during multicompoenting In terms of the Amplification plot the baseline is a line fit to defined range of cycles before the sequence detection system detects the amplification of the PCR product For standard PCR assays the SDS software uses a default range of cycles 3 15 to establish the baseline for a PCR assay In relative quantification analysis a sample to which all other samples are referenced on a detector by detector basis A DNA molecule with a nucleotide sequence that is complementary to an RNA
180. cal Adjustment r of Text box arrows 1 step per click 4 Check the rotary position of the Plate Handler arm to confirm that the gripper e is centered over the stack will not contact the sides of the stack when lowered 7 42 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Aligning the Plate Handler 5 Using the Rotary Adjustment arrows adjust the rotational position of the gripper so that it is centered over the input stack and will not contact the sides when lowered e To move the Plate Handler arm clockwise click the up arrow e To move the Plate Handler arm counter clockwise click the down arrow Rotary Positioning RA Up arrow moves the arm clockwise pe 04 Down arrow moves the arm counter clockwise 6 Using the Vertical Positioning commands carefully lower the Plate Handler arm into the stack Adjust the Rotary Adjustment value as needed to center the gripper inside the stack 7 After the gripper is centered inside the stack click Find Plate f The Plate Handler arm lowers upon the plate Confirm the following e the plate is in the middle of the gripper span e the plate sensor switch is contacting the plate e the gripper and plate do not contact the side of the stack 8 Click Close Gripper The gripper grips the plate between its fingers 9 Select WW Vertical Home The Plate Handler raises the arm to its highest position If
181. cation In Process Citation Electrophoresis 20 2 291 299 1999 Lo Y M et al Quantitative analysis of fetal DNA in maternal plasma and serum implications for noninvasive prenatal diagnosis Am J Hum Genet 62 1998 768 775 Lo Y M Chan L Y Lo K W Leung S F Zhang J Chan A T Lee J C Hjelm N M Johnson P J and Huang D P Quantitative analysis of cell free Epstein Barr virus DNA in plasma of patients with nasopharyngeal carcinoma Cancer Res 59 6 1188 1191 1999 Lo Y M Leung T N Tein M S Sargent I L Zhang J Lau T K Haines C J and Redman C W Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia see comments Clin Chem 45 2 184 188 1999 Lo Y M L Y Chan A T Chan S F Leung K W Lo J Zhang J C Lee N M Hjelm PJ Johnson and D P Huang 1999 Quantitative and temporal correlation between circulating cell free Epstein Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma Cancer Res 59 5452 5455 Leutenegger C M Klein D Hofmann Lehmann R Mislin C Hummel U Boni J Boretti F Guenzburg W H and Lutz H Rapid feline immunodeficiency virus provirus quantitation by polymerase chain reaction using the TaqMan fluorogenic real time detection system In Process Citation J Virol Methods 78 1 2 105 116 1999 Lockey C E Otto and Z Long Real time fluorescence detection of a single DNA molecule BioTechniques 24 19
182. cessfully integrating the Applied Biosystems 7900HT Fast Real Time PCR System into a laboratory workflow During a single 24 hour period of real time operation the 79700HT instrument can produce more than 200 MB of data To successfully manage the information produced by the 7900HT instrument you should have a basic understanding of how data is collected stored and processed throughout the operation of the instrument The Applied Biosystems 7900HT Fast Real Time PCR System has three standard modes of operation depending on the accessories purchased with the base instrument e Stand Alone Operation see below e Automated Operation see page 1 18 e Database Operation see page 1 18 Stand Alone Operation This mode uses the most basic configuration of the Applied Biosystems 7900HT Fast Real Time PCR System Figure 1 11 where the 7900HT instrument is used without any additional components In this configuration a technician runs plates individually using the SDS software which stores all data on the computer hard drives Computer SDS Software Plate Document Created Applied Biosystems 7900HT Hard drive s Instrument Firmware to Run a Plate Fast Real Time PCR System Plate Document Used Thermal Cycling and Sequence Detection Raw Data Saved to the Plate Document Data Collection Plate Document Opened and Analyzed Figure 1 11 Stand Alone Opera
183. change from the drop down list then click ox e Enter a description of the reason for the change in the field then click Reasons for change drop down list Resson For Change Atribute AGPlate cu veue New valje Default descriptions Calculation Error Meed to Change Threshold Erie ihe Reaconior Change Meed to Reanalyze Example of a custom description for a change Figure 2 4 Reasons for Change Dialog Box Options Custom description of the reason for the change Electronic The Electronic Signature Verification dialog box appears only if the SDS Enterprise Signature Database is configured to require electronic signatures The electronic signature Verification system restricts and tracks user access of the SDS data contained by the database Dialog Box If the Electronic Signature Verification dialog box appears 1 In the User ID and Password fields enter your user name and password then click oK 2 After the software validates your user name and password it displays a message stating the success or failure of the signature Click x to continue Description of the action that requires a signature ti n You attempted is crontroller by electronic signature Please enter you usert name and password Co proceed Enter your user name Enter your password Figure 2 5 Electronic Signature Verification Dialog Box Options Applied Biosystems 7900HT F
184. ck or select File gt New Pure Dye Plate Document Start the SDS software Configure the New Document dialog box Assay Select Pure Spectra Container Select the appropriate format Template If performing a e Standard 384 Well Pure Dye Run select 384 Well Pure Dyes Plate sdt If no plate document templates are available construct a Pure Dye plate document using the product insert from the Spectral Calibration Kit and the procedure on page 7 28 e Standard 96 Well Pure Dye Run select the plate document template matching the Pure Dye plate you intend to run To run Plate 1 containing FAM JOE NED and ROX dyes select 96 Well Pure Dyes Plate 1 sdt To run Plate 2 containing SYBR TAMRA TET and VIC dyes select 96 Well Pure Dyes Plate 2 sdt 7 22 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Performing a Pure Dye Run e Fast 96 Well Pure Dye Run select the plate document template matching the Pure Dye plate you intend to run To run Plate 1 containing FAM JOE NED and ROX dyes select Fast 96 Well Pure Dyes Plate 1 sdt To run Plate 2 containing SYBR TAMRA TET and VIC dyes select Fast 96 Well Pure Dyes Plate 2 sdt e Microfluidic Pure Dye Run leave the field blank To run the microfluidic pure dye card containing FAM dye select MFC FAM Pure Dyes sdt To run the microfluidic pure dye card containing ROX dye select MFC ROX Pure Dyes s
185. cks the file preventing any changes from being made to it until the plate document is run or removed from the queue Note To release the plate document from the queue start the Automation Controller Software and remove the plate document from the queue as explained on page 4 40 5 Click OK 6 Select File gt Close The SDS software closes the plate document 7 Repeat the procedures in this chapter to create plates and add plates to the queue as needed 8 When you finish creating plate documents run the queued plates as explained in Starting and Configuring the Automation Controller Software for Operation on page 4 39 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 35 Chapter 4 Operating the Instrument Creating Plate Documents Using the Template Batch Utility About the The Template Batch utility allows you to quickly create multiple plate documents Template Batch from a single SDS 7900HT Template Document sdt The Template Batch utility Utility can be a useful timesaving device in situations where samples are run on plates with identical assay configurations IMPORTANT Plate documents created by the Template Batch utility do not contain sample or plate information Bar code or Comment settings You must apply this information to each plate document individually after the file is run Generating Plate Note For more information on the elements of the Template Batch d
186. closing 2 18 configuring document information 3 25 copying detectors 3 11 copying markers 3 13 creating 2 13 3 8 creating from a template 3 24 4 36 exporting 2 14 importing setup data 2 16 A 2 methods See methods opening 2 19 programming methods 3 17 removing from the plate queue 4 40 running batches 4 39 running individually 3 26 saving 2 17 saving as a single plate file 4 24 saving as a template file 3 22 saving to the database 4 24 setting Sample Volume 3 20 setting the passive reference 3 16 plate grid about 2 29 selecting wells 2 24 viewing well information 2 23 zooming 2 26 Plate Handler 1 8 7 36 adjustment knob 7 36 aligning 7 41 to 7 48 cleaning the finger pads 7 52 gripper 7 36 plate stack positions 7 36 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Index 5 plate sensor switch 7 36 replacing the finger pads 7 52 turning ON 2 4 plate queue adding plate documents Automation Controller Software 4 40 adding plate documents SDS software 4 35 adding plate documents Template Batch utility 4 36 removing plate documents 4 40 starting 4 43 stopping 4 44 plate stacks loading plates 4 41 4 42 placing in use 4 42 positions 7 36 Plateau Phase of the amplification curve D 5 plates running batches 4 39 4 41 running individually 4 25 types See consumables plate sensor switch 7 36 adjusting 7 37 to 7 40 plots hiding 2 21 maximizing minimizing 2 22 resizing 2 21 2
187. ct File gt Open In the Look in field navigate to and select the plate document file Click on The SDS software displays the plate document file Configure the Analysis Settings for each marker as explained below Opening Un analyzed Data from the SDS Enterprise Database 5 6 Click or select File gt Open Document from Database In the Plate Query dialog box configure the Find Plates Matching These Criteria table with parameters that correspond to the session of interest Click Search In the Search Results list scroll to and select the session that you want to analyze Click open 10 Click _ bene when finished Analyzing the Run Data User Access Requirement Analyzing the Run There is no access requirement All users can analyze dissociation curve data that has been saved to the SDS Enterprise Database The run data from a temperature ramp can be analyzed immediately following the completion of the run For an explanation of how the software manipulates the raw data see Algorithmic Manipulation of Raw Data on page 6 7 l 3 Select all wells in the plate grid The software outlines the selected wells with a black border Choose one of the following e Select Analysis gt Analyze e Click jp The software analyzes the run data and displays the results in the Dissociation Curve tab Determine the T for the dissociation curves displayed within the Dissociati
188. d Curve Method 6 3 troubleshooting 8 12 removing plate documents from the plate queue 4 40 steps from a method 3 19 Rep readout from the Real Time tab 4 27 replacing gripper finger pads 7 52 sample block module 7 6 replicate wells use of 6 20 reproducible limits 6 19 resizing panes views and plots 2 21 restacking plates 4 42 restoring database connection A 22 RQ Manager Software 1 25 RQ Min Max Confidence 6 28 running batches of plates 4 39 4 41 single plate 4 25 S sample calibrator 6 20 sample bars about 6 31 Index 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide sample block locking bar 7 8 sample block locking bolt 7 8 sample block module about 1 5 cleaning sample block module wells 7 15 contamination 8 7 replacing 7 6 sample names adding to a plate document 3 25 importing into a plate document A 2 Sample readout from the Real Time tab 4 27 sample volume setting 3 20 saturation signal 7 27 saving plate documents 2 17 4 24 template files 3 22 scrutinizing allele calls 5 16 SDS 7900HT Document sds files See plate documents SDS 7900HT Template Document sdt files See templates SDS Enterprise Database about 1 22 command line tools 1 26 RQ Manager Software 1 25 saving plate documents 4 24 saving results 5 19 6 53 saving templates 3 23 SDS Database 1 26 SDS User Account Manager 1 27 SNP Manager Software 1 25 study centric design 1 27 SD
189. d Query Results Click to begin Search Results the search Seale ae 6 31 PM an Kesici orat ee a a O eT 3BaN00B9v2 6 6 02 9 38PM Relative Quantific EFAULT meet the search Open Select All Deselect All Clear All criteria appear here Dane In the Search Results list scroll to and select the document that you want to analyze Click open When the software has loaded the plate document click _ bone Configure the Analysis Settings for each marker as explained in Configuring the Analysis Settings on page 6 11 6 10 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Analyzing the Data Analyzing the Data User Access There is no access requirement All users can analyze absolute quantification data Requirement that has been saved to the SDS Enterprise Database Configuring the Before analyzing the plate document data you must configure the analysis settings Analysis Settings for each detector that you want to analyze The analysis settings are detector specific and must be set for each one individually For each detector the SDS software can calculate baseline and threshold values automatically using the AutoC algorithm or manually using values you supply Note You can analyze the run without configuring the analysis settings however the software will analyze the data using a default baseline of cycle 3 to 15 and a default threshold value of 0 2 1
190. d all applicable Material Safety Data Sheets MSDSs See About MSDSs on page xviii Neo PHYSICAL INJURY HAZARD Use this instrument as specified by Applied Biosystems Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide xvii Safety and EMC Compliance Information Chemical Safety Chemical Hazard ANITY CHEMICAL HAZARD Before handling any chemicals refer to Xvili Warning About MSDSs Obtaining MSDSs the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions N VEGANI CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument Nore CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Nore CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking
191. d from a standard TaqMan RNase P Instrument Verification Plate The arrangement of the samples and standards on the plate are shown in Examples in This Chapter on page 6 8 a Curve ee c SS Sannin Unknowns Quantity LogN initial concentration Figure 6 3 Standard Curve Generated from a TaqMan RNase P Instrument Verification Plate 6 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Overview Algorithmic The SDS software can analyze raw data immediately after an absolute quantification Manipulation of run is complete The term raw data refers to the spectral data between 500 nm to Raw Data 660 nm collected by the software during the real time run During the analysis the software automatically applies several mathematical transformations to the raw data to generate a more direct measure of the relationship between the spectral changes in the unknown samples Multicomponenting The first mathematical transformation involves the conversion of the raw data expressed in terms of Fluorescent Signal vs Wavelength to pure dye components using the extracted pure dye standards At the same time the software determines the contribution of each dye in the raw data using the multicomponent algorithm Setting the Threshold and Calling C s After normalization the baseline and threshold values must be set for the run see Kinetic Analysis Quantitative PCR on page D 4 for more inform
192. d inside the dialog box or window in which you are working For end point applications such as allelic discrimination the throughput of the Applied Biosystems 7900HT Fast Real Time PCR System can be increased by dividing the workload between the 7900HT instrument and several thermal cyclers Unlike real time runs the 7900HT instrument collects data for end point runs after the completion of the PCR Consequently you can perform the thermal cycling of end point plates elsewhere and then transfer them to the 7900HT instrument afterwards for data collection and analysis IMPORTANT To perform the thermal cycling and the plate read using the 7900HT instrument run the plate first as a real time plate document and then again as an allelic discrimination plate document see Step 5 Programming the Plate Document Method on page 3 17 for the procedure Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 3 Chapter 3 Preparing a Run Workflow Overview Experiments Runs Performed on the 7900HT Instrument Absolute Quantification Workflow Absolute Quantification sses dite ods taanads en bad nbre ieee d Naa 3 4 Allelic Discrimination ss ssererssescsisrericse terest iesto drsti 3 6 Backo OU 4 25 net eee eee EErEE ee ede eee eee CA See Fee eens Ta 7 16 Dissociation Curve Melting Curve 00 00 cece eee eee 3 5 Pure Dye Spectral Calibration 0 0 0 00 000 000 ccc eens 7 20
193. data collection parameters e Stores data gathered during the run SDS 7900HT sdm amp e Used to analyze relative quantification data Multiple Plate Hi small studies Document SDS 7900HT sdt r e Used to create plate documents Template _ e Can store assay data thermal cycling and data Document collection parameters Imported Exported Files Assay Plate txt Used to create plate documents Document Setup Files Exported Data txt e Contains exported raw or analyzed data for all or Files a select group of wells on a plate document e Uses txt a tab delimited format compatible with most spreadsheet applications JPEG Graphic jpg t e Most panes and plots of the plate document can File be exported as JPEG graphic files e Compatible with most word processing and spreadsheet applications e Can be incorporated directly into HTML or XML documents The use of plate document templates is optional but useful as a timesaving device for experiments where samples are run on plates with identical assay configurations tJoint Photographic Experts Group tlcon will varies depending on the application associated with the image file type Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 15 Chapter 1 Product Overview Plate Document File Size The SDS software can produce plate document files of varying sizes depending on the type of runs fo
194. ded by Applied Biosystems To add custom pure dyes to the Pure Dye set for your instrument see Adding Custom Dyes to the Pure Dye Set on page 7 27 7 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Performing a Pure Dye Run After a run the SDS software receives run data in the form of a raw spectra signal for each reading To make sense of the raw data the software must determine the contribution of each fluorescent dye used in the sample through a process called multicomponenting The software accomplishes the separation by comparing the raw spectra with a set of pure dye standards contained in a calibration file When a plate document is saved after analysis the software stores the pure spectra information with the collected fluorescent data for that experiment Materials 384 or 96 Well Plate Required e Appropriate Spectral Calibration Kit Sequence Detection Systems 384 Well Spectral Calibration Kit ABI PRISM 7900HT Sequence Detection Systems 96 Well Spectral Calibration Kit 7900HT System Fast 96 Well Spectral Calibration Kit Note Both the standard 96 well and Fast 96 Well Spectral Calibration Kits contain two pure dye plates e Product Insert e Centrifuge with plate adapter Microfluidic Pure Dye Cards e Microfluidic card spectral calibration reagents e Centrifuge e Microfluidic card sealer Applied Biosystems 7900HT Fast Real Time PCR System and SDS
195. des of the optical adhesive covers the heat from the lid may bake the liquid to the cover and may distort the signal If splashing occurs briefly centrifuge the reaction plate to remove all traces of liquid from the caps 8 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Low Precision or Irreproducibility Drops Drops of reagents that cling to the sides of the wells may not contact the thermal cycler sample block and consequently may not amplify If the drop slides into the mix during PCR then the amplified products will become diluted and the result will be less than replicate wells that did not have drops Therefore carefully monitor the reaction plate as it is being transferred into the thermal cycler or 7900HT instrument If you observe any drops take steps to remove them such as centrifugation Writing on the Do not write on any surface of the optical plates or the optical adhesive covers The Reaction Plates fluorescent properties of the ink can potentially affect the fluorescence emission from the plate and alter the results Instead note the contents of each well on a sheet of paper or on a printout of the sample setup Fluorescent Many compounds found in laboratories are fluorescent If they come into contact Contamination on _ certain optical surfaces such as the optical adhesive covers the fluorescent results the Plates may be affected For example it has been noted that the powder u
196. djusting analysis options for absolute quantification 6 11 display settings 3 22 method step parameters 3 19 plate sensor switch 7 37 to 7 40 adjustment knob 7 36 air bubbles 8 6 aligning fixed position bar code reader 7 49 to 7 51 Plate Handler 7 41 to 7 48 allele calls about 5 15 calling 5 14 scrutinizing 5 16 allelic discrimination about 5 6 to 5 8 analyzing run data 5 10 to 5 18 assay development guidelines B 5 maximizing throughput 3 3 procedure checklist 5 10 thermal cycling on the 7900HT instrument 3 18 troubleshooting 8 13 Allelic Discrimination Plot about 5 13 calling alleles 5 14 datapoint cluster variations 5 8 exporting as a graphic file A 16 exporting data as a text file A 16 genotypic segregation 5 8 outliers 5 8 scrutinizing allele calls 5 16 amplification beyond reproducible limits in multi reporter experiments 6 19 amplification curve about D 4 Geometric Exponential Phase D 4 Linear Phase D 5 Plateau Phase D 5 Amplification Plot exporting as a graphic file A 16 exporting data as a text file A 16 Analysis 6 22 analysis sessions about 1 28 analyzing absolute quantification data 6 9 to 6 13 allelic discrimination data 5 10 to 5 18 background data 7 19 dissociation curve data 6 40 to 6 44 pure dye data 7 25 relative quantification data 6 22 to 6 35 applying detector tasks 3 14 detectors to a plate document 3 11 markers to a plate document 3 13 sample names 3 25 architecture database LAN set
197. dt To run the microfluidic pure dye card containing VIC dye select MFC VIC Pure Dyes sdt 4 In the Barcode field scan the bar code number using the hand held bar code reader 5 Click The software displays a plate document with the attributes for a pure dye run 6 Save the Pure Dye plate document a b C d Click J or select File gt Save Select Files of type gt SDS 7900HT Document sds In the File name field if running a Standard 384 Well Plate enter PureDye_ lt date in MMDDYY format gt For example the file name for a plate run on May 31 2001 would be PureDye_053101 Standard 96 Well Plate enter PureDye_Plate lt plate gt _ lt date in MMDDYY format gt For example the file name for a Pure Dye Plate 1 run on May 31 2001 would be PureDye_Plate1_053101 Fast 96 Well Plate enter FastPureDye_Plate lt plate gt _ lt date in MMDDYY format gt sds For example for a fast pure dye plate 1 run on May 31 2001 type FastPureDye_Plate1_053101 sds Microfluidic pure dye card enter lt Dye gt lt date in MMDDYY format gt _MFC For example the file name for a run performed on May 31 2001 would be FAM_053101_MFC Click seve The software saves the plate document 7 Prepare and run the pure dye plate or card as explained below Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 23 Chapter 7 Maintaining the Instrument Running th
198. e 11 Configure the setup table file with plate document information detector task marker and sample data as explained on page A 3 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Importing Plate Document Setup Table Files Configuring the Setup Table File with Plate Document Information The second step in the procedure is to import the setup table file into a secondary application configure it with sample and detector information and then save the completed setup table file in tab delimited format l 2 Start the application that you want to use to edit the setup table file Import the setup table file from the previous procedure as tab delimited text If using a spreadsheet application to edit the setup table file the application automatically parses the tab delimited information into the cells of a spreadsheet Configure the setup table file with sample and detector information according to the file structure explained on page A 4 Save the setup table file in tab delimited format Import the completed setup table file into an empty plate document as explained below Importing the Completed Setup Table File into a Plate Document The final step in the procedure is to import the completed setup table tab delimited file into an empty plate document l If the plate document created in Creating an Empty Setup Table File on page A 2 is still open in the SDS softwa
199. e e Sorvall Heraeus Tool less 750 mL Swing Out Rotor Body e A set of Sorvall Heraeus Custom Buckets four each with its own card holder Contact your Applied Biosystems representative to order the custom buckets e Current firmware If you are upgrading an existing Sorvall Legend T or Legend RT Centrifuge you may also need to upgrade the firmware so that it can recognize the Low Density Array bucket type Centrifuge Figure 4 8 illustrates the centrifuge system components System Components SC Ceentrifuge rotor Provides the rotational drive for a set of four centrifuge buckets Card holder Supports the Low Density Array s fill reservoirs during centrifugation Bucket Contains the Low Density Arrays and card holder during centrifugation Figure 4 8 Centrifuge System Components 4 12 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Laboratory Setup Setting the Bucket Type for the Low Density Array Preparing TaqMan Low Density Arrays for Use The centrifuge system is designed to be placed on a standard laboratory bench It requires access to 115 VAC 60 Hz electrical power Consult the owner s manual should for specific operating and site preparation instructions 1 Power on the centrifuge 2 Following the instructions in the Sorvall Legend T Centrifuge operator s manual use the front panel controls on the centrifuge to set the bucket type fo
200. e For more information on adjusting the appearance of the plot click the help button and see the Sequence Detection Systems Software Online Help e Step drop down list Chooses the data displayed within the plot based on the ramp If a plate document contains data from more than one temperature ramp the Step drop down list allows you to displays the data from each by selecting the position of the ramp in the thermal profile 6 42 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Determining T Values for the Analyzed Run e T display and slider The SDS software displays the T below the green slider see below TT 90 0 86 5 Tm There are two definitions for the T value The chemical definition is the temperature at which 50 of the DNA is ina double stranded configuration The mathematical definition is the maximum value for the first derivative curve within a specific peak e Plot drop down list Chooses the data displayed within the plot based on the derivative calculation The list offers the following selections Raw When selected this option plots the normalized reporter fluorescence data R on a graph of fluorescence vs temperature left most plot in Figure 6 16 Derivative When selected this option plots derivative data R on a graph of the derivative vs temperature right most plot in Figure 6 16 The derivative data
201. e document until you are comfortable using the feature 2 22 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Basic Software Skills Tutorial Lesson 3 Using the Plate Grid Overview The plate grid see Figure 2 6 is an important interface tool for the SDS software The software uses the grid to convey information about the plate and allows you to select specific wells for viewing and analysis The following exercises will teach you how to use and modify the elements of the plate grid Plate grid Figure 2 6 Plate Grid of SDS Plate Documents Exercise 1 The SDS software provides two methods for viewing the information associated with Viewing Well a well or wells of the plate document To view the information for a well of the plate Information document do one of the following e Click any well in the plate grid to select it When selected the software outlines the well in black and displays the associated information in the well inspector of the Setup tab Setup tab displays the information of the selected well ot Bi ae raion a Passe Py crane a Fi bce PB pane Be Fie Hib T Bir T a a E a H asses iy cna Maie i r Baie HE yi mi T mf af j e Move the pointer over any well of the plate grid The software displays the information for the well in a yellow pop up window aed oad ae eo ad ae oor aad a ee eee ee ee Ss a Pop up displays the information
202. e 7900HT instrument for use with optical plates of the same format 384 Well 96 Well or 96 Well Fast The TaqMan RNase P Instrument Verification Plates are pre loaded with the reagents necessary for the detection and quantification of genomic copies of the human RNase P gene a single copy gene encoding the RNase moiety of the RNase P enzyme Each well contains pre loaded reaction mix master mix RNase P primers and FAM labeled probe and template Table 7 2 illustrates the arrangement of standards and samples on each type of TaqMan RNase P Instrument Verification Plate As shown below the TaqMan RNase P Instrument Verification Plates consist of five columns of template standards 1250 2500 5000 10 000 and 20 000 copies and two unknown populations 5000 and 10 000 copies Table 7 2 Configurations of the TaqMan RNase P Instrument Verification Plates TaqMan RNase P Instrument Verification Sample Configuration Plate TaqMan RNase P 384 Well Instrument Verification Plate PN 4323306 ROL QEQ0 A OCOCOC00 GR2107 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Purpose of TGF B Runs Materials Required Verifying Instrument Performance Table 7 2 Configurations of the TaqMan RNase P Instrument Verification Plates TaqMan RNase P Instrument Verification Sample Configuration Plate TaqMan RNase P Instrument Verification Plate POSO OOTO ace
203. e Positions of the Zymark Twister Microplate Handler Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 4 Chapter 4 Operating the Instrument Loading Plates 1 Following the guidelines on page 4 41 load the sealed plates or cards into the Plate Handler stacks In the Automation Controller Software select the check boxes for the plate stacks containing plates IMPORTANT If you are not using stack 1 or the Restack option explained below remove all plates from stack 1 before starting the queue Under these settings the Plate Handler will attempt to stack the run plates from stack 2 in the stack 1 position If stack 1 contains plates these settings will cause the Plate Handler to stop the run Ii SDS Automation Controller Of x Fie Instrument Enterprise Help Plate Queue Thermal Status Plate Name Run Type C Program Files 4pplied Biosystems S C Program Files Applied Biosystems C Program Files 4pplied Biosystems S C Program Files Applied Biosystems C Program Files Applied Biosystems C Program Files 4pplied Biosystems S C Program Files Applied Biosystems S C Program Files Applied Biosystems C Program Files 4pplied Biosystems S C Program Files Applied Biosystems C Program Files Applied Biosystems S C Program Files 4pplied Biosystems S C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program File
204. e Prepared Pure Dye Plate or Card Running the 1 Load the pure dye plate or microfluidic pure dye card into the 7900HT Pure Dye Plate or instrument Card a In the plate document in the SDS software select the Instrument tab b In the Real Time tab in the Instrument tab click Spenjclose The instrument tray rotates to the OUT position c Place the pure dye plate or card into the instrument tray as shown below Well A1 Position the plate so that the bar code faces towards the front of the instrument Well A1 Position the microfluidic pure dye card so that the bar code faces towards the front of the instrument Note The A1 position is located in the top left side of the instrument tray 2 Click San The 7900HT instrument begins the pure dye run The method for a pure dye run is coded into the software and consists of a single 2 min hold at 60 C Note Before starting the run the instrument may pause up to 15 min to heat the heated cover to the appropriate temperature 3 When the pure dye run is complete and the Run Complete dialog box appears a Click ok to close the dialog box b Click openictose and remove the pure dye plate or card from the instrument tray c Extract the pure dye calibration information as explained on page 7 25 7 24 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Performing a Pure Dye Run Analyzing the Pure
205. e Sample well assignments e Marker detector definitions and well assignments e Marker detector tasks and quantity e Plate bar code Analysis Sessions An analysis session 1s a set of analysis results derived from a single raw data set associated with a single plate document analyzed by the SDS software Multiple analysis sessions can be associated with the same plate document Each analysis session has a unique name used to distinguish it from the other sessions for the same plate document Session data exists in two states on the database attached or unattached Attached sessions are those that have been assigned to a specific study Session data can be attached to a study by the Automation Controller Software or by using the RQ Manager or SNP Manager Software IMPORTANT After you attach a session to a study you cannot add it to another study until you unattach it See the RO Manager Software User Guide or SNP Manager Software User Guide for instructions on removing attached sessions from a study RQ and SNP Studies The SDS Enterprise Database software uses a system of studies to organize the secondary analysis of session data produced by an Applied Biosystems 7900HT Fast Real Time PCR System The central purpose of using the study system is to group the analysis session data for analysis using the SNP Manager or the RQ Manager Analysis Software Supporting API Documentation Technology Support The SDS Enterprise Database inc
206. e Size 384 Output Plate ID 384N75822034 xxx Number of Detectors 2 Detector Reporter Quencher CYP 2C9 2 1 FAM CYP 2C9 2 2 VIC xxx Number of Markers 1 Marker Allelex AlleleyY CYP 2C9 2 CYP 2C9 2 2 CYP 2C9 2 2 Well Sample Name Detector Task 1 Sample 1 2 Sample 2 3 eer 384 Sample 48 Well Sample Name Marker Task 1 Sample 1 CYP 2C9 2 UNKN 2 Sample 2 CYP 2C9 2 UNKN 3 alien 384 Sample 48 CYP 2C9 2 UNKN Description PDAR CYP 2C9 2 All PDAR CYP 2C9 2 Al2 Description PDAR CYP 2C9 2 Quantity Marker Task Comments Example Probe Example Probe Comments Example marker Example Code A 13 Absolute quantification setup table files xxx SDS Setup File Version 3 Output Plate Size 96 Output Plate ID 96JG729FF903 Number of Detectors 1 Detector Reporter Quencher Description Comments RNase P FAM TAMRA RNase P Probe Example Probe Well Sample Name Detector Task Quantity Detector Task Quantity 1 Sample 1 GAPDH UNKN 0 2 Sample 2 GAPDH STND 20000 3 Poea 96 Sample 30 GAPDH UNKN 0 Example Code A 14 Relative quantification setup table files SDS Setup File Version 3 Output Plate Size 96 Output Plate ID 96FRE505SDA2 Number of Detectors 2 Detector Reporter Quencher Description Comments GAPDH VIC GAPDH Probe Example Probe TNF Q FAM TNF Probe Example Probe Well Sample Name Detector Task Quantity Detector Task Quantity 1 Calibrator GAPDH ENDO 0 TNF Q UNKN 0 2 Sample 1 GAPDH ENDO 0 T
207. e Zymark Twister Software can cause portions of the program to persist in memory even after the software has been closed Because the Zymark Twister Software conflicts with the SDS software these residual elements must be closed inside the Windows Task Manager before continuing Confirm that the Zymark Twister Software has closed by viewing the Task Manager a Press the Ctrl Alt Del keys in unison b In the Windows Security dialog box click Task Manager c In the Task Manager dialog box confirm that the Twister software has closed by looking for the Twister software entry in the Task list If the software is still running click the software entry and click End Task to exit the software d Select File gt Exit to quit the Task Manager Replace the covers for the fixed position bar code reader and the underlying platform removed in step 1 on page 7 41 Fixed position bar code reader and underlying platform covers 7 48 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Aligning the Fixed Position Bar Code Reader Aligning the Fixed Position Bar Code Reader Description The fixed position bar code reader must be set so that it automatically scans the plate s bar code as the plate is placed into the instrument tray by the Plate Handler Preparing the 1 Remove the cover for the fixed position bar code reader Instrument for the Alignment
208. e can conduct comparative analyses of over 97 detectors 96 target genes and 1 endogenous control across 200 plates or approximately 153 600 multiplexed reactions run on an Applied Biosystems 7900HT Fast Real Time PCR System instrument See the RO Manager Software User Guide PN 4351670 for more information Creating the Study Creating a Multiple Plate Document Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide To conduct a comparative analysis of a series of relative quantification plates in a study you must first create a Relative Quantification Multiple Plate Document 1 In the SDS software click j or select File gt New 2 Configure the New Document dialog box with the following settings e Assay Relative Quantification AAC Study e Container the plate formats used to run the samples in the study IMPORTANT Relative quantification studies cannot compare data from runs performed using different plate formats for example samples run in a 96 well plate cannot be compared to those run in a 384 well plate 3 Click __ The software displays a new plate document with the appropriate attributes 4 Add the relative quantification plate documents to the study as explained in Adding Plate Documents to the Study on page 6 24 6 23 Chapter 6 Analyzing Real Time Data Adding Plate Documents to the Study To conduct a comparative analysis of a series of relati
209. e can handle multiple documents simultaneously the processing speed of the computer decreases with each open document Consequently Applied Biosystems recommends limiting the number of open documents to 10 Figure 2 8 shows a typical plate document and identifies its components which are described on the following pages Tabs Plate grid see below fe sps 2 1 olx Fie Edt View Tools Instrument Analysis Window Help DSUs F P RS 4 s el TA a E Ma Ma M m e a Rj 96 Well RNaseP Install Plate sdt Absolute Quantification fe Nese 2s a e eee oe Me ese toe as Ae a EHREREEEHEEEE SE E Mee o GA o Oe ee o GA ee o GA o Gi q i PIES gij Sample Name M RNaseP FAM Detector Reporter Task Quantity Color B Disconnected n GA GD Gs DA GA GA s GA s GA lt DA s GA GAl s G SEB RR Bee eee s DA GA s GAI s DAI GA s DA s GA s GAI s DA s GA s GAl s G z EE BEER EEE o Gii o GAl o GA o GAl o GA o GA o GA o GA o GA o GA o GA o G E E E E E E E E E E_E o Gai o GAl o GA o GAl o GA o GA o GA o GA o GA o GA o GA o G TE E E E E E E_E _ E_E Oe o GA o GA o GA o GA o GA o Oe o GA o GA o GA ee al gt F Ts Position Sample Detector Task ct Qua 41 SK RNaseP Unknown a 42 SK RNaseP Unknown 43 SK RNaseP Unknown Add Detector Clear Gopy to Manager 44 SK RNaseP Unknown a 45 SK RNaseP Unknown Passive Reference Rox 4
210. e caution AA caution Indicates that you should Indicates the presence of moving parts and to proceed with appropriate caution Indicates the presence of an electrical shock hazard and to proceed with appropriate caution Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution gt gt amp Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide XV Safety and EMC Compliance Information Safety Labels on Instruments The following CAUTION WARNING and DANGER statements may be displayed on Applied Biosystems instruments in combination with the safety symbols described in the preceding section English Francais CAUTION Hazardous chemicals Read the Material Safety Data Sheets MSDSs before handling ATTENTION Produits chimiques dangeureux Lire les fiches techniques de s ret de mat riels avant la manipulation des produits CAUTION Hazardous waste Read the waste profile if any in the site preparation guide for this instrument before handling or disposal ATTENTION D chets dangereux Lire les renseignements sur les d chets avant de les manipuler ou de les liminer CAUTION Hazardous waste Refer to MSDS s and local regulations for handling and disposal ATTENTION D chets dangereux Lire les fiches techniques de suret de materiels et la regulation locale associ es a
211. e computer or displays an error message e Incorrect start up sequence e Corrupted software e Computer hardware failure e Operating System OS corruption e Loose bar code reader cable Follow the solutions listed until the symptom goes away 1 1 Power off the 7900HT instrument 2 Check cable connections 3 Restart the computer and logon to the computer 4 Power on the 7900HT instrument 5 Start the SDS software 2 6 Restart the computer and logon to your computer 7 Reinstall the SDS software 8 Start the SDS software 3 Contact Applied Biosystems Service for OS problems or if the computer will not boot up at all You may have to reload the OS from the CDs 4 Contact Dell for troubleshooting the computer hardware Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Software and 7900HT Instrument Table 8 4 Troubleshooting Software and Computer Problems Observation Possible Cause Recommended Action Communication error Cables are connected incorrectly Check cable connections and COM port setup See Instrument Connections on page 1 10 Thermal cycler errors Sample block module not fully engaged Reseat the sample block module as explained Replacing the Sample Block on page 6 Automation Controller Software cannot find a plate document file File not in correct
212. e document template 4 Configure the new plate document with sample names and plate information as explained on page 3 25 3 24 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Step 8 Applying Sample and Plate Information Step 8 Applying Sample and Plate Information User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to apply or modify sample names to the plate document Applying Sample The plate document must contain sample attributes to effectively organize and Names to the analyze data produced from the run Once applied the software displays the sample Plate Document names in the plate grid and table views Note You can apply sample names after the plate has been run but they must be added before the analysis of the run data Note The SDS software provides the ability to import setup table information detector detector task and sample name layouts into a plate document from a tab delimited text file See Importing Plate Document Setup Table Files on page A 2 for more information 1 In the plate grid select the wells containing the first sample 2 Click the Sample Name field enter a name for the sample and press Enter The software labels the selected wells with the new sample name 3 Repeat steps through 2 for all remaining samples D RNaser P RNaser Dy auacer
213. e from the following If running a single plate then continue to step 2 If running the first plate in a series of plates with identical assay configurations then save the plate document as a template see page 3 22 6 Create a plate document from the template created in step 2 see page 3 24 7 Configure the document with sample names and plate information see page 3 25 a Prepare the allelic discrimination plate or plates and perform thermal cycling on a designated thermal cycler see page 4 1 b Run the allelic discrimination plate or plates on the 7900HT instrument 8 Analyze the run data see page 5 5 Steps 2 and 2 can be eliminated by importing the plate document setup information from a tab delimited text file See Importing Plate Document Setup Table Files on page A 2 for more information 3 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Quick Review Powering On the 7900HT Instrument Quick Review Powering On the 7900HT Instrument Powering On the Instrument Note The following table includes a set of abridged procedures for activating the components of the Applied Biosystems 7900HT Fast Real Time PCR System For a complete explanation of the procedure see Powering On the 7900HT Instrument on page 2 4 IMPORTANT Do not power on the 7900HT inst
214. e injury illness or death JN Nail les CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical Waste e Safety Guidelines Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying the waste container s
215. e plate document template Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 37 Chapter 4 Operating the Instrument 10 Repeat the procedures in this Chapter 3 Preparing a Run and Chapter 4 Operating the Instrument to create and add additional plates to the queue as needed Note After you have added a plate document to the plate queue the software locks the file preventing any changes from being made to it until the plate document has been run or removed from the queue 11 When finished creating and adding plate documents to the queue run the queue as explained in Starting and Configuring the Automation Controller Software for Operation on page 4 39 4 38 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Operating the Software without an SDS Enterprise Database Starting and Configuring the Automation Controller Software for Operation The Applied Biosystems 7900HT Fast Real Time PCR System employs the Automation Controller Software for automated operation of the 7900HT instrument The software coordinates the action of the 7900HT instrument the bar code reader and the Plate Handler while acquiring and saving raw data during each run Starting and Configuring the Software 1 Ifrunning the SDS software do one of the following e Select Instrument gt Disconnect to discontinue communication between the software and the in
216. e plate document with any additional custom dyes Setup instrument Dyes Custom 01 Dyes drop down list Custom dye added to selected wells of the plate document 5 Save the custom Pure Dye plate document as a plate document template a Click or select File gt Save b In the Save dialog box navigate to Program Files gt Applied Biosystems gt SDS 2 2 1 gt Templates The Templates directory appears inside the Look in field By saving the plate document template to the Templates directory it becomes available from the Template drop down list in the New Document dialog box c Select Files of type gt SDS 7900HT Template Document sdt d In the File name field and enter a name for the plate document template e Click swe The software saves the plate document as a plate document template 6 Run the custom Pure Dye plate as explained in Preparing the Pure Dye Plates on page 7 22 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 29 Chapter 7 Maintaining the Instrument Verifying Instrument Performance When to Perform Applied Biosystems recommends performing an RNase P or TGF B run 30 Purpose of RNase P Runs e When changing sample block formats for the first time e As needed to verify the function of the 7900HT instrument The TaqMan RNase P Instrument Verification Plates are experiments run to verify the performance of th
217. e plate stacks differently than the Automation Controller Software Figure 7 6 lists the positions defined by the Zymark Twister Software and the Automation Controller Software equivalents f Automation Zymark Twister Controller O W Software Software 2 Position 0 Output G i Position 1 unused Position 2 Instrument Position 3 unused front of instrument Bar code Well A1 Position 4 Stack 1 Figure 7 6 Positions of the Automation Controller and Zymark Twister Software 7 36 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Adjusting the Sensitivity of the Plate Sensor Switch Adjusting the Sensitivity of the Plate Sensor Switch When to Perform Materials Required Adjusting the Plate Sensor Switch The plate sensor switch located underneath the arm of the Zymark Twister Microplate Handler requires adjustment under the following circumstances e When changing sample block module formats e Ifthe Plate Handler is having difficulty sensing plates e Optical plate of the current sample block format The dimensions of different plate formats can place different requirements on how the Plate Handler grips plates To ensure smooth operation of the Automation Accessory adjust the plate sensor switch when changing consumable formats 1 Power off the Zymark Twister Microplate Handler A ANETA PHYSICAL HAZARD The Zymark Twister Microplate
218. e the AutoC algorithm Applied Biosystems recommends that you verify that the baseline and threshold values are set correctly for each detector configured for AutoCalling see page 6 46 6 26 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Analyzing the Study e Manual Ct Instructs the software to use the baseline and threshold values specified in the Analysis Settings dialog box to calculate C s for the selected detectors The software provides two methods for manually setting the baseline and threshold values for the detector If you know the values that you want to use enter them into the appropriate fields of the Analysis Settings dialog box If you want to set the baseline and threshold using the controls of the Amplification Plot tab leave the fields empty and set the values later as described on page 6 46 Replicate and The settings of this group box determine how the software identifies and removes Outlier Removal outlier data for all detectors in the study for Study Settings A Replicate and Outlier Removal Automatic Outlier us it Automatic Outlier Removal Removal check box a Omit replicates whose ACE lt f o imultiplex onl Omit replicates whose AC lt check box Figure 6 10 Replicate and Outlier Removal for Study Settings e Automatic Outlier Removal Instructs the software to automat
219. e the data needed to create a curve the 7900HT instrument performs a programmed temperature ramp in which it slowly elevates the temperature of the plate over several minutes The specific binding characteristic of the SYBR Green 1 Dye permits the 7900HT instrument to monitor the hybridization activity of the nucleic acids present in the sample During the run the instrument records the decrease in SYBR Green fluorescence resulting from the dissociation of dsDNA After the run the SDS software processes the raw fluorescence data from the SYBR Green Dye to generate a more meaningful representation of the relationship between spectral change and temperature for the dissociation curve run Multicomponenting and Normalization The first mathematical transformation involves the conversion of the raw data expressed in terms of Fluorescent Signal vs Wavelength to pure dye components using the extracted pure dye standards At the same time the software determines the contribution of each dye in the raw data using the multicomponent algorithm Afterwards the software normalizes the data using the component of the passive reference dye as shown below R SYBR R Oe R PassiveRefercnce Derivation of Dissociation Curve Data The SDS software then computes the first derivative of the normalized data R for each reading taken by the 7900HT instrument during the temperature ramp The resulting derivative data R is the rate of
220. e to the skin and can cause skin depigmentation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Nee CHEMICAL HAZARD Isopropanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin and cause irritation It may cause central nervous system effects such as drowsiness dizziness and headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves This procedure applies to the sample block on the 7900HT System TaqMan Low Density Array Upgrade 1 Wipe the surface of the sample block grey aluminum with 10 bleach 2 Wipe the sample block three times with distilled water 3 Wipe the sample block with isopropanol then allow to air dry v s A Circuitry and connections to A the instrument Do Not Touch SS Well A 1 4 Replace the block as explained in Replacing the Sample Block on page 7 10 5 Runa background Low Density Array to confirm that the contamination has been removed Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Decontaminating the Sample Block Cleaning a This procedure applies to a Sample Block Standard 384 Well Block Standard 96 Well Block F
221. e unique and should reflect the target locus of the assay such as GAPDH or RNase P Do not use a name for more than one detector The SDS software does not distinguish between detectors of the same name even if they use a different dye set Group field Optional Enter or select a detector group for the detector Description field Optional Enter a brief description of the assay up to 32 characters Reporter Quencher drop down lists Select the appropriate reporter and quencher dyes for the probe If creating a detector for an assay using the SYBR Green dsDNA Binding Dye set the Quencher Dye drop down list to Non Fluorescent If you are using a custom dye not manufactured by Applied Biosystems you must create and run a pure dye plate for the dye before applying it to a detector see Adding Custom Dyes to the Pure Dye Set on page 7 27 Color box Optional Click the box then use the Color Picker dialog box to select a color to represent the detector and click ox Notes field Optional Enter any additional comments for the detector up to 200 characters Name field Group field Description field Marne Group Meronen Reporter Quencher drop down lists Quencher Mon Fluorescent coor ll Color box Motes Notes field Last Modified Oct 10 2003 11 36 02 AM Oct 10 2003 11 36 02 AM Created Creation and Modification Applied Biosy
222. e using the hand held bar code reader Click __ The software creates a plate document Note Do not modify the plate document The plate document template is pre programmed with detector and method information for the run Save the plate document a Click or select File gt Save b In the Barcode field of the Save dialog box do one of the following Enter a name or bar code number for the plate then click sse Using the hand held bar code reader scan the bar code number c Select Files of type gt SDS 7900HT Document sds d Click _ se The software saves the plate document The software is now configured for the RNase P run Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 33 Chapter 7 Maintaining the Instrument Running the Verification Plate or TGF B Card Verifying Instrument Performance 1 In the plate document in the SDS software select the Instrument tab 2 In the lower portion of the Instrument tab select the Real Time tab 3 Ifthe instrument tray is inside the 7900HT instrument click Open close The instrument tray rotates to the OUT position 4 Place the TaqMan RNase P Instrument Verification Plate or TGF B card into the instrument tray Note The A1 position is located in the top left corner of the instrument tray 5 In the Real Time tab click Start The 7900HT instrument begins the run Note Before starting the PCR run t
223. eRe SARREN eee ess 7 12 Decontaminating the Sample Block s40 7425 94044406404045 hers ceed oun 7 14 Performing a Background Run 0 eens 7 16 Pertonning a Purs Dye Run 9 4c 0554 hd oesaind r oi bn0S bo eee ee ees doew es 7 20 Adding Custom Dyes to the Pure Dye Set 0 0 0 0 00 ccc e es 7 27 Verifying Instrument Performance 0 teens 7 30 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 5 Chapter 7 Maintaining the Instrument Replacing the Sample Block When to Perform Sample Block Installation Workflow IMPORTANT Before changing the sample block perform all required upgrades to the SDS software and instrument firmware Failure to update the software can make the instrument inoperable or result in damage to instrument components You need to remove the 7900HT instrument sample block when you e Decontaminate the wells of the sample block see page 7 14 e Change sample block formats IMPORTANT Always run a background plate after installing the sample block Unless instructed to do otherwise adhere to the following guidelines when exchanging sample block modules of different formats To install a sample block module 1 Perform all required software and firmware upgrades to the Applied Biosystems 7900HT Fast Real Time PCR System see page 7 55 Remove the existing sample block see page 7 7 Install the new sample block see page 7 10 Change the
224. eal Time PCR System and SDS Enterprise Database User Guide 7 25 Chapter 7 Maintaining the Instrument b Ifthe data set contains an outlying peak eliminate it by clicking the check box of the associated well Note Dye spectra are generally acceptable if they peak at the same location as their group but diverge slightly at other wavelengths c Click Net when finished d Repeat steps a to c for all remaining wells until prompted with a message reporting the extraction of the pure dyes The software extracts the pure spectra and stores the data as a component of the calibration file 3 Click J or select File gt Save The software saves the plate document 4 Select File gt Close The software closes the plate document 5 If performing spectral calibration of a e Standard 384 Well Block The pure dye calibration is complete e Standard 96 Well Block Run the second Pure Dye plate by repeating the procedures for Preparing a Plate Document for a Pure Dye Plate or Card on page 7 22 Running the Prepared Pure Dye Plate or Card on page 7 24 Analyzing the Pure Dye Run on page 7 25 e 7900HT System TaqMan Low Density Array Upgrade Run the second and third microfluidic pure dye cards containing the VIC and ROX dyes by repeating the procedures for Preparing a Plate Document for a Pure Dye Plate or Card on page 7 22 Running the Prepared Pure Dye Plate or Card
225. eal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide XIX Safety and EMC Compliance Information Waste Profiles Waste Disposal A waste profile for the Applied Biosystems 7900HT Fast Real Time PCR System is provided in the Applied Biosystems 7900HT Fast Real Time PCR System Site Preparation Guide Waste profiles show the percentage compositions of the reagents in the waste stream generated during installation and during a typical user application even though the typical application may not be used in your laboratory The waste profiles help you plan for the handling and disposal of waste generated by operation of the instrument Read the waste profiles and all applicable MSDSs before handling or disposing of chemical waste If potentially hazardous waste is generated when you operate the instrument you must e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory e Ensure the health and safety of all personnel in your laboratory e Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT R
226. ector tasks as explained in Step 3 Configuring the Plate Document with Tasks on page 3 14 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 13 Chapter 3 Preparing a Run Step 3 Configuring the Plate Document with Tasks User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to apply or modify detector task settings About Detector You must assign a task to the detectors or markers applied to each well of the plate Tasks document that defines their specific purpose or function on the plate The SDS software uses the detector task assignments to determine how to treat the data produced by the wells when analyzing the run data Detector tasks vary depending on the type plate document Applying 1 Using the Ctrl and Shift keys select the wells of the plate grid containing Detector Tasks samples for a particular task described in the table below Table 3 1 Detector Tasks of the SDS Software Experiment Task Apply to Allelic Unknown all detectors of wells that contain PCR reagents Discrimination and test samples NTC all detectors of negative control wells that contain reagents for the PCR but lack template Absolute Unknown all detectors of wells containing PCR reagents and Quantification test samples for quantification Standard the appropriate detectors of wells that contain PCR rea
227. ed Using the Rotary Adjustment arrows adjust the rotational position of the gripper so that it is centered over the input stack and will not contact the sides when lowered Using the Vertical Positioning commands carefully lower the Plate Handler arm into the stack Adjust the Rotary Adjustment value as needed to center the gripper inside the stack After the gripper is centered inside the stack click Find Plate f The Plate Handler arm lowers upon the plate Confirm the following e The plate is in the middle of the gripper span e The plate sensor switch is contacting the plate e The gripper does not contact the side of the stack Click lose Gripper The gripper grips the plate between its fingers Select Yertical Home The Plate Handler raises the arm to its highest position If the plate contacts the sides of the stack re adjust the rotary position of the Plate Handler arm until the plate moves freely in the stack Note Contact between the plate and the stack may be unavoidable However try to minimize the contact as much as possible Using the Vertical Positioning commands raise and lower Plate Handler arm several times to check the alignment Lower the Plate Handler arm and click Rota Orset and click _ tes The software records the rotary position for position 0 the output stack 7 46 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 13 14 15 16
228. ed Biosystems C Program Files Applied Biosystems S C Program Files Applied Biosystems C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program Files Applied Biosystems S Instrument Control S84N00LQ04 S84NO0LA00 S84NO0LO S84N00LO02L 384NO00LR 3A S84NO0LRA5 S84NO0LRADS 384NO08HKE 384N0257M0 384N00LQQC S84NO0LA IX 384NO0LQRD S84N025075 S84NO0LON 384N016N9 Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED DONE DONE Plate Queue must contain plate documents for all plates to be run Robot s Active Stacks Open Close IV Stacki 7 Stack2 Stack3 7 Stack 4 M Restack when finished Studies Save Allelic Discrimination to Study Example Study Save Relative Quantification to Study a Ready 16 plate s Online 6 Prepare and load the plates onto the Plate Handler as explained on page 4 41 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 39 Chapter 4 Operating the Instrument Adding Plates to the Plate Queue NO e In the Automation Controller Software select File gt Add Plates In the Look in field of the Open dialog box navigate to the directory conta
229. ed detectors To determine the baseline and threshold values Automatically Select the Automatic Ct option button See page 6 18 for information on the Automatic Ct algorithm If you choose to use the Automatic Cy algorithm verify that the baseline and threshold were called correctly for each detector configured for AutoCalling following the procedure on page 6 46 Manually Do the following a Select the Manual Ct option button b In the Threshold field enter a threshold value to apply to the selected detector s or leave the field empty and set the threshold value manually as explained on page 6 46 c Select one of the following e Automatic Baseline to have the software calculate a baseline for the selected detector s e Manual Baseline then enter Start and End cycle values to apply to the selected detector s e Manual Baseline and leave the Start and End cycles fields empty You will need to set the values manually as explained on page 6 46 4 If necessary repeat steps 2 through 3 for any additional detectors IMPORTANT The threshold and baseline values set in step 3 are detector specific and must be set for each individual detector used on in the plate document 5 If evaluating groups of two or more replicates per sample select or deselect the Automatic Outlier Removal option 6 If desired select the Omit Samples Whose AC lt check box then enter a value in the field See Replicate and Outl
230. ed within the dialog box or window in which you are working Examples in This The screenshots that appear within this chapter were created using a series of Chapter TaqgMan Cytokine Gene Expression Plates PN 4304671 a research tool for real time in vitro quantitative evaluation of human cytokine gene expression The plate detects the expression of 12 cytokine target sequences and an endogenous control in complementary DNA cDNA samples The TaqMan Cytokine Gene Expression Plate I consists of a MicroAmp Optical 96 Well Reaction Plate arranged into 24 replicate groups each containing TaqMan probes and primers for the assay of one human cytokine mRNA and an endogenous control Figure 6 8 illustrates the assay configurations for the plate The study used to produce the screenshots for this section consisted of four plates of a time course experiment run to evaluate the expression of 24 target cytokine mRNA over a 36 hour period 36 hr O 1 O 2 O 3 O 4 o 5 O 6 O O 8 O 9 o 10o 11 O 12 38 OBOHOOCSOOO GOO SOAOQOO ONOOBOO oun sO OOOO O00 OOOO en ee eee Plate 2 Plate 2 00 0D 9UIR CGV0 O FO00000000000 60 00 000000000 D 1 1 640 000000000000 h eles A OB O O D O Omes O a G o Mash E GND O O TO j PITT TIT IIT on 10 B OO OOOO BOO emea 0 0 OO OO OO OO OO OO
231. elect the session that you want to analyze Click oen Click bene when finished Configure the Analysis Settings for each marker as explained below Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 5 11 Chapter 5 Analyzing End Point Data Analyzing an Allelic Discrimination Run User Access Requirement About the Analysis Configuring the Analysis Settings Analyzing the Data There is no access requirement All users can analyze allelic discrimination data The analysis of SNP or genotyping data involves the automatic or manual calling of sample data for each marker The calls are data labels assigned to individual samples to reflect their genomic content The SDS software can call sample data The SDS software can call sample data e Manually using the toolbar and scatterplot e Automatically using the AutoCalling system see About the AutoCalling System on page 5 7 Before analyzing your data you must configure the analysis settings for each marker that you want to analyze using the AutoCalling algorithm The analysis settings are specific to each marker and must be set for each one individually IMPORTANT You need to configure the analysis settings for a marker only if you want to use the AutoCalling system to analyze it To configure the analysis settings 1 Click or select Analysis gt Analysis Settings 2 Do one of the follo
232. emperature Time Remaining 00 01 Sample 25 0 Block 24 9 Data Collection Stamp Cover 89 4 Pre Cycle Post Stage 1 Step 1 Time 00 00 Rep 1 State Not Started Real Time Tab Plate Read Tab Real Time Runs End Point Runs Figure 4 10 Real Time and Plate Read Tabs of the SDS Software e Status Displays the condition of the 7900HT instrument e Time Remaining Displays the calculated time remaining in the run Temperature group box Real Time Plate Documents Only e Block Displays the actual temperature of the sample block module e Cover Displays the actual temperature of the heated cover e Sample Displays the calculated temperature of the samples Cycle group box Real Time Plate Documents Only e Rep Displays the current cycle repetition e Stage Displays the current stage of the thermal cycling e State Displays the current condition of the cycle stage e Step Displays the current step being run e Time Displays the calculated time remaining in the current step Data Collection Stamp group box End Point Plate Documents Only e Pre Displays the date that the instrument performed the pre read e Post Displays the date that the instrument performed the post read Note A pre read is a plate read performed before a plate has undergone thermal cycling Note A post read is a plate read performed after a plate has undergone thermal cycling Note For more information
233. en display temperatures Yes No the installation is successful The presence of temperature readings confirm that the 7900HT instrument successfully established the connection to the new sample block the 7900HT instrument is unable to establish communication with the new sample block To troubleshoot the problem 1 Power off and unplug the 7900HT instrument 2 Remove the thermal cycler access cover 3 Press on the right and left sides of the front plate of the sample block to ensure that it is seated securely 4 Reinstall the thermal cycler access cover 5 Repeat step 8 until you hear a high pitched tone confirming communication between the instrument and sample block 10 After the sample block is loaded into the instrument do the following a Perform a background run see page 7 16 to verify that the sample block Is connected and working properly Contains no contaminants that will interfere with fluorescent detection If changing sample block formats perform any remaining tasks outlined in the Sample Block Installation Workflow on page 7 6 IMPORTANT Perform pure dye and background runs after replacing the sample block even if you are replacing a sample block module with another block of the same format Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 11 Chapter 7 Maintaining the Instrument Changing the Plate Adapter W
234. ent as a Template Saving the Plate IMPORTANT Saving the plate document as a plate document template is an optional Document as a __ Step and recommended for instances where the document can be used to create Template duplicate plate documents for a series of plates with identical assay configurations If Optional you choose not to use your plate document as a plate document template go to Configuring the Plate Document Information Optional on page 3 25 To save the plate document as an SDS 7900HT Template Document l 2 Select File gt Save As In the Save in field of the Save As dialog box navigate to the AppliedBiosystems gt SDS2 2 1 gt Templates directory Note By saving the file to the Templates directory it becomes available from the Template drop down list in the New Document dialog box Select File of type gt SDS 7900HT Template Document sdt Click the File name field and enter a name for the plate document template Click save The software saves the plate document template If the software displays one or more warnings read the note and click Select File gt Close If the software prompts you to save the plate document click The SDS software closes the plate document template Create a plate document from the plate document template as explained on page 3 24 To save the plate document template to the SDS Enterprise Database 1 Select File gt Save Template to Database
235. entify the components of the amplification curve and set the threshold so that it is e Above the background e Below the plateaued and linear regions e Within in the geometric phase of the amplification curve e Irregular amplification Do all samples appear to have amplified normally The three phases of the amplification curve should be clearly visible in each signal e Outlying amplification When the run data is viewed in the Cy vs Well Position plot do replicate wells amplify comparably Wells producing Cy values that differ significantly from the average for the associated replicate wells may be considered outliers If a plate produces non uniformity between replicates some samples on the plate could have evaporated Check the seal of the optical adhesive cover for leaks End Point Runs Allelic Discrimination Troubleshooting When faced with irregular data you can use the SDS software to diagnose some Analyzed Data chemistry and instrument related problems The following table contains a summary of checks for verifying the integrity of your run data and to help you begin troubleshooting potential problems Raw Data The Raw Data Plot displays the raw reporter fluorescence signal not normalized for the selected wells during each cycle of the PCR What to look for e Signal tightness and uniformity Do the raw spectra signals from replicate groups and controls exhibit similar spectral profiles If not the plate
236. eparing 7 32 T table pane about 2 29 exporting A 16 TaqMan fluorogenic probe about D 2 designing B 2 to B 3 TaqMan Low Density Array centrifuging 4 16 components 4 10 handling 4 14 how it works 4 10 loading 4 14 loading guidelines 4 14 number of targets per well 4 10 overview 4 10 placing in bucket 4 16 quality control 4 12 removing from packaging 4 14 sealing 4 19 Index 7 single plex TaqMan reagents 4 11 TaqMan primers and probes 4 10 trimming after sealing 4 21 TaqMan reagents description 4 10 TaqMan RNase P Instrument Verification Plates about 7 30 analyzing 7 34 kits C 5 preparing a plate document 7 33 running 7 30 7 32 Targets mRNA number per well 4 10 tasks See detector tasks templates about 2 12 creating a single plate document 3 24 creating multiple plate documents 4 36 saving as 3 22 thermal cycler block See sample block module thermal cycling protocol See methods threshold about D 6 configuring for automatic analysis 6 11 improper setting 8 5 threshold cycle calculation D 6 relationship to PCR product D 7 Time readout from the Real Time tab 4 27 Time Remaining readout from the Real Time tab 4 27 Tm See melting temperature troubleshooting 8 2 to 8 20 7900HT instrument 8 14 background runs 8 9 chemistry problems 8 5 to 8 8 computer 8 14 end point runs 8 13 fixed position bar code reader 8 17 8 20 pure dyeruns 8 11 real time runs 8 12 SDS software 8 14 Zymark Twister Microplate Ha
237. eporter fluorescence even after cleavage 9 Select probes with a Primer Express software estimated T of 65 67 C 3 Make the TaqMan MGB probes as short as possible but no fewer than 13 nucleotides in length Avoid runs of an identical nucleotide This is especially true for guanine where runs of four or more should be avoided Position the polymorphic site in the central third of the probe Note The polymorphic site can be shifted toward the 3 end to meet the above guidelines however the site must be located more than two nucleotides upstream from the 3 terminus The following figure illustrates the placement of a polymorphism in an example probe N Nucleotide First try to position the polymorphic Do not place site in the central third of the probe it here 5 3 N NNNNNNNNNNNNNNNNNNNN Polymorphism If necessary place the polymorphism here B 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Design Tips for Quantitative PCR Assays Design Tips for Quantitative PCR Assays Selecting an Selecting a good amplicon site ensures amplification of the target mRNA without Amplicon Site for co amplifying the genomic sequence pseudogenes and related genes Gene Expression Applied Biosystems recommends the following guidelines when selecting an Assays amplicon site for quantification assays e Primers and probes must be designed following the A
238. er Finger Pads on page 7 52 Gripper pads are worn or dirty Change the gripper pads as explained in Cleaning and Replacing Gripper Finger Pads on page 7 52 Restack when finished option not selected Configure the Automation Controller Software to restack the plates as explained in page 4 42 Bar code reader is misaligned Bar code reader is broken Re align the fixed position bar code reader as explained in Aligning the Fixed Position Bar Code Reader on page 7 49 Chapter 8 Troubleshooting TaqMan Low Density Array Low Density Array Table 8 6 Troubleshooting the TaqMan Low Density Array Troubleshooting Table Observation Possible Cause Recommended Action After removing the Low N A If the fill consumable is Density Array from its damaged then the PCR packaging the fill reaction mixture might not consumable is damaged flow into the reaction wells creased bent or folded during centrifugation Discard the Low Density Array After removing the Low N A If the aluminum foil backing Density Array from its is damaged then the PCR packaging the aluminum reaction mixture might not foil backing is damaged flow into the reaction wells creased bent or folded during centrifugation Discard the Low Density Array After removing the Low N A Remove dust or particulates Density Array from its packaging dust or other particulates settle on the reaction wells
239. er if the data from the allelic discrimination run produces unexpected results 6 When the run is complete close the absolute quantification plate document 7 Go on to Step 6 Saving the Plate Document as a Template on page 3 22 3 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Programming Methods for Absolute or Relative Quantification Step 5 Programming the Plate Document Method Note The following procedure describes how to configure only the basic features of the method thermal cycler conditions sample volume and data collection options To further customize the method for the plate document click in the Instrument tab and refer to the online help for instructions on configuring the auto increment and ramp rate values l 2 In the SDS software select the Instrument tab of the plate document If necessary select or de select the 9600 Emulation check box Note When the 9600 Emulation check box is checked the SDS software reduces the ramp rate of the 7900HT instrument to match that of the ABI PRISM 7700 Sequence Detection System instrument Modify the default thermal profile for the method as needed To Then adjust step select a field value enter a new value and click anywhere parameters outside of the field time temp add a hold cycle set or step Stage 3 Repeats lao E 0 E 0 00 Temperature field 4 to
240. erivation of the AAC Equation see ABI PRISM 7700 Sequence Detection System User Bulletin 2 Relative Quantification Of Gene Expression PN 4303859 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 17 Chapter 6 Analyzing Real Time Data Automatic Threshold and 6 18 Baseline Determination The SDS software features a proprietary AutoC algorithm that can be used to automatically generate baseline and threshold values for individual detectors The algorithm calculates baseline and threshold parameters for a detector based on the assumption that the data exhibits the typical amplification curve shown in Figure 6 7 2 000 E 1 1 000 E 1 Plateau phase Linear phase Geometric phase 2 166 E 1 Background i ry Foi a N A 4 te f i H KA ARN VAR AAE 0 a 6 8 10 12 14 is 18 20 24 26 28 30 32 34 36 38 4 Baseline Cycle Figure 6 7 Typical Amplification Curve Experimental error such as contamination pipetting inaccuracy and so on can produce amplification curves that deviate significantly from the typical data shown above The data from these irregularities can affect the AutoC algorithm by causing it to generate incorrect baseline and threshold parameters for the associated detector For this reason Applied Biosystems strongly recommends reviewing all baseline and threshold parameters determined by the AutoC algorithm foll
241. erprise Database User Guide Basic Software Skills Tutorial Exercise 4 The Save command stores any changes to the plate document setup information and Saving a Plate display settings If using the version of the SDS software that includes the client Document software for the SDS Enterprise Database you also have the option of saving the plate document file to the database Choose from the procedures below e Ifusing a database save the plate document to the database as described on page 2 18 e Ifyou are not using a database save the plate document to your computer hard drive as described below Saving the Plate Document as an SDS 7900HT Document 1 Click J or select File gt Save As 2 Select File of type gt SDS 7900HT Document sds 3 In the File name field enter Practice E save As Ei Look in SD5 Documents E File name Practice sds Save Files of type aer Prism 505 Document sds Cancel 4 Click Save k 5 In the Document not properly set up warning click Document not properly set up x AN This plate does not contain any detector information It cannot be analyzed until detectors are defined and added to wells The software saves the plate document to a file entitled Practice sds 6 Close the saved plate document file as explained on page 2 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 17 Chapter 2 Getting St
242. erprise Database User Guide V Chapter 6 vi Analyzing Real Time Data Notes for Database Users 0 ccc eee teens 6 2 Real Time Runs on the 7900HT Instrument 2 0 00 ees 6 3 Section 6 1 Absolute Quantification 0 ce n nnn nn nananana 6 5 OVEINICW 2 ket EAEE NEN ohn howe pee ees ae ee oan eee ke 6 6 Before YOU BEGIN ers Sunes ete ea eee ees Set GS Rae net eae ee eae Ae ee aa 6 8 Aialysis GneCKliSt ics 2 ci 4 tence gr tite an Oe he ew hate ee ar ad ae ear pane ewes pee 6 9 Opening the Run Datars sestta ses outs beeen Be eee Bae ees 6 10 Anay Nge Asi ie ht ws eek leat Boe td Sate tera he aed rd a a et Sie vey el deed tana Bele eal 6 11 VIGWINIGHPCSUILG aaga amp 8k dre dre ele seated ome A at oh eee ated tate eee oa E aN a NA 6 13 After TAC ANGIVSIS i ww of et ana a Me ecg ae eat Ge hom a ean Ms ace Boa ak ad RE are es 6 14 Section 6 2 Relative Quantification 0 0 0 es 6 15 OV EIMIGW ss leicester Sneed nde a deed ae ee tae ws ar Shae oh ae i 6 16 Algorithmic Manipulation of Raw Data 0 0 ee 6 17 Automatic Outlier Removal 0 0 0 0 ce eee eee 6 19 Essential Experimental Components 000 00 ccc eee ee ee eee 6 20 Genad lt 5 2 0 ness eaten oine es eee sd eee ee eee aged aa seaet ees 6 21 Arialysis GNCCKIISE a 74 2 Sih gehen ieee te Clea Se he aed gS hee ed 6 22 Options for Analyzing Relative Quantification Data 0 0 eee eee 6 23 reatingo Ne Stuy tra see
243. es When one template is diluted Concentration several times as with the RNase P target in the RNase P Instrument Verification and Cycle Plates see Appendix C the ratio of template concentration to detectable signal is Number Preserved in the exponential phase for all dilutions see Figure D 4 This relationship appears to change as rate of amplification approaches a plateau 20000 10000 5000 2500 1250 Cycle Number Figure D 4 Amplification Plot from a Real Time Run of an RNase P Instrument Verification Plate Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide D 5 Appendix D Theory of Operation Fluorescence vs Amplified Product Calculating Threshold Cycles When using TaqMan fluorogenic probes with the 7900HT instrument fluorescence emission increases in direct proportion to the amount of specific amplified product As Figure D 4 on page D 5 demonstrates the graph of normalized reporter R vs cycle number during PCR appears to have three stages Initially R appears as a flat line because the fluorescent signal is below the detection limit of the 7900HT instrument In the second stage the signal can be detected as it continues to increase in direct proportion to the increase in the products of PCR As PCR product continues to increase the ratio of AmpliTagq Gold polymerase to PCR product decreases When template concentration reaches 10 8 M PCR product ceases to gro
244. expression within which the true expression level value is likely to fall The software calculates the error bars based on the RQMin and RQMax confidence interval setting in the Analysis Settings dialog box Note Since standard deviation can only be calculated from two or more replicates the SDS software does not display error bars RQMin and RQMax values or standard deviation if either the target or the endogenous control has only a single sample Note Error bars are a representation of the likelihood of a random sample meeting acceptance criteria based on standard of population degrees of freedom and RQ Min Max confidence values The software does not display error bars for the calibrator sample because it is compared to itself Undetermined The software declares sample as undetermined if the amplification curves for the Samples and associated replicates in the group do not cross the threshold for the detector during Outlier Removal the duration of the run e If outlier removal is not turned on then the software ignores any undetermined wells and displays the Cy of the remaining wells as the average Cy e If outlier removal is turned on then voting occurs If the majority of the wells in the sample replicate group are undetermined then the software displays the average Cy as undetermined If half or more of the wells have valid C values then the software ignores the undetermined wells removes outliers from the remaining wells
245. f Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4351684 Rev C 06 2010 Contents Preface How to Use This Guide 0000 cee ee eee eee ee es How to Obtain More Information 0 00 ee ee ee es How to Obtain Services and Support naaa 0 00 eee Safety and EMC Compliance Information Safety Conventions Used in This Document 0 000 e eee eee SYMDOlS OniINSruIMENIS yu oe eae ee dee See 2 alo eae o Safety Labels on Instruments 0 00 cece eens General Instrument Safety 0 0 0 00 ccc eee eens Chemical Saleh c c254 5 cae AREE ada wk been 6 pee AAA REREN aiei Chemical Waste Safety 0 00 cc eee ene Elec IC Al ode ae Fong nance usenet ets ee eee eee eae oe meet Physical Hazard Safety escapa So WG Gee hd be as ei SI ew ek es Biological Hazard Safety censas iceri terba ee ees Laser Sally aww ane ths ok yg el wade gs oda eed eo A aaa Anal Ge ara Bar Code Scanner Laser Safety 000 0 eee eens WOrKStallon oale atic ty deh ca te Rae Soe ee ole Seow ee eid ae ee ee Safety and Electromagnetic Compatibility EMC Standards Chapter 1 Product Overview SY Sle OVERVIEW soo iene hha ath Anke ee aw we ee a ace tae ea a ed Section 1 1 Getting to Know the Hardware 00 ce eee TIO ASTUME 2 621 28864 ureednocaacect 4 ohee kee eich oan oo Goede COMDPULEF kersare se ha ai A ees et de eet COG A oa
246. f absolute quantification is to accurately determine the absolute quantity of a single nucleic acid target sequence within an unknown sample The results of an absolute quantification experiment are reported in the same unit of measure as the standard used to make them Absolute quantification on the 7900HT instrument is accomplished through the use of the polymerase chain reaction and the fluorogenic 5 nuclease assay see page D 2 During setup standards diluted over several orders of magnitude and unknown samples are loaded onto an optical plate containing master mix and TaqMan reagents targeting a specific nucleic acid sequence The plate is then loaded into a 7900HT instrument which has been configured to perform a real time run During the thermal cycling the instrument records the emission resulting from the cleavage of TaqMan probes in the presence of the target sequence After the run the SDS software processes the raw fluorescence data to produce threshold cycle C values for each sample see page D 6 The software then computes a standard curve from the Cy values of the diluted standards and extrapolates absolute quantities for the unknown samples based on their C values see below Note See Appendix D Theory of Operation for more information on the fluorogenic 5 nuclease assay real time data collection or the mathematical transformations of sequence detection data Figure 6 3 illustrates a standard curve generate
247. f at all times during the following procedure Failure to comply can result in serious physical injury to the user or damage to the instrument 4 Wait 20 to 30 min for the heated cover to cool ASNrEe PHYSICAL HAZARD During instrument operation the temperature of the sample block can be as high as 100 C Before performing this procedure wait until the sample block reaches room temperature Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 7 Chapter 7 Maintaining the Instrument 5 Ifthe instrument tray is in the OUT position outside of the instrument push it into the instrument to provide an open workspace 6 If using a Zymark Twister Microplate Handler remove the covers for the fixed position bar code reader and the underlying platform Fixed position bar code reader and underlying platform covers 7 Push the instrument tray inside the instrument then remove the thermal cycler access cover to permit access to the sample block IMPORTANT The thermal cycler access cover is secured to the instrument by non locking pins and may require force to remove it no tools are required Access cover 8 Using a 5 16 inch hex key turn the sample block locking bolt counter clockwise until it is very loose but still attached to the sample block locking bar IMPORTANT Some instruments may require the use of an adjustable crescent wrench to lo
248. f major procedures described inside this manual Setting Up and Running SDS Experiments Creating an SDS Plate Document 2 2 04 44 4444444 atstat t aN 4os eee bees 3 8 Running an Individual SDS Plate Document 0 0 0 0 eee 4 23 Running Batches of SDS Plate Document 00 000 cae 4 31 Stopping a Run from the SDS Software 0 000000 0c 4 28 Stopping a Run from the Automation Controller Software 4 4 Fjecting a Plate from the SDS Software 0 0 eee 4 29 Ejecting a Plate from the Automation Controller Software 4 4 Analyzing Run Data Analyzing an Allelic Discrimination Run 0 0 0 0 00 eee 5 5 Analyzing an Absolute Quantification Run 0 0 0 cece eee 6 5 Analyzing an Relative Quantification Run 0 0 0 ene 6 15 Analyzing a Dissociation Curve Melting Curve Run 00 6 37 Maintaining the 7900HT Instrument Changing the Plate Adapter 2 sci dacckd evens deeb gee eew hed de eeeee hes 7 12 Kepigcimg ihe Sane IOC nd ctcedewanadde de dean deeue ee 4485 oe eEe es 7 6 Decontaminating the Sample Block 0 0000 0 cece ees 7 14 Performing a Background Run 0 000 7 16 Performing a Pure Dye Run ccs ands ec can hed d bande ee ea we neusdiownds aden 7 20 Adding Custom Dyes to the Pure Dye Set 0 0 000s 7 27 Verifying Instrument Performance Using a TaqMan RNase P Instrument WORM cota teta
249. f target sequences in the unknown samples The expression of a target gene relative to control gene in one sample relative to the expression level in a calibrator sample See AAC After plates have been loaded into the Zymark Twister Microplate Handler stacks and the Automation Controller Software has been told to start running no human intervention should normally be required until all plates in the robot stacks have been processed With the SDS Enterprise Database option this includes plate document analysis and analysis results persistence to the database Describes the ability to analyze a large quantity of samples for genotyping or gene expression analysis A thermal cycling set point of a method that maintains a specified temperature for an extended period of time The physical location of a site on a chromosome sometimes understood as the site of a gene usually understood as the site of a marker variant For SDS software a virtual representation of a variable segment of DNA such as a SNP whose inheritance can be followed comprising a name and a set of two detectors specific for the allelic variants Markers are used only for allelic discrimination assays not for absolute quantification or relative quantification Allelic discrimination plate documents must contain at least one marker The marker must be configured with two detectors before it can be applied to a plate document The protocol that the Applied Biosystems 790
250. false the software hides the document from view and excludes the data from the analysis As explained later in this section the Search tool can evaluate multiple expressions many rows to refine the data set Search Statement Figure A 1 shows a filter created in the Add Files dialog box that illustrates the basic Syntax structure of all statements Logic Operator Column Category Conditional Operator Value Criteria A logical operator Describes the category Describes the natureof Contains the condition IF AND or OR for the evaluation the evaluation for the evaluation E add Plates Plates To Be Added To Study C pplied Biosystems SDS Docume C pplied Biosystems SDS Docume C pplied Biosystems SDS Docume C pplied Biosystems SDS Docume File Search Search in eiea Bi T Include Subdirectories jects Matching This Criteria tee coum coronan veue fee feras a a A Search Clear i Append Results Search Results Existing Plates In The Study ICL ait EDS Documents ihe Clear All j Murmber of Plates Study can hold 10 6 free OK Rene Figure A 1 Add Files dialog box A 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Using the Search Tool Logical Operators Every statement begins with one of three logical operators IF OR or AND The IF AND OR logical operator determines the relationship be
251. fication Plate consists of 5 columns of template standards 1250 2500 5000 10 000 and 20 000 copies and two replicate populations 5000 and 10 000 copies 15 16 718192022234 O10 OO COO0 00 olexorexerexorere O10 OO C0 0000 00 009000000 Gere C16 Sxelore 5 Q Population 2 or O 10000 OOM WW YY x OOOO S010 09 O 0 0 0 0 0 0 OO OOOCCO000 Population 1O 5000 LZY ZV Y O LO IN T fa H N a aeheatiey chs Si ceeececdet doped ROCCO DO Clbpb bbb OO GO 0000 GR2107 Figure 6 4 Sample Configuration of the Example Plate 6 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Analysis Checklist Analysis Checklist Workflow Overview 1 Open the plate document See page 6 10 2 Configure the analysis settings for the run see page 6 11 v 3 Analyze the run data see page 6 12 4 For each detector that you configured to determine the baseline and threshold values see page 6 46 Automatically Verify that the baseline and threshold values for the detector were set correctly by the software Manually Set the baseline and threshold values for the detector Y 5 Visualize outliers and eliminate any outlying amplification from the run data see page 6 49 6 View the results of the absolute quantification run see page 6 14 7 Choose
252. fluorescence by the reporter After thermal cycling the plate is run on the 7900HT instrument which reads the fluorescence generated during the PCR amplification By quantifying and comparing the fluorescent signals using the SDS software it 1s possible to determine the allelic content of each sample on the plate Mismatches between a probe and target reduce the efficiency of probe hybridization Furthermore AmpliTag Gold DNA polymerase is more likely to displace the mismatched probe than to cleave it releasing the reporter dye By running the extension phase of the PCR at the optimal annealing temperature for the probes the lower melting temperatures T for mismatched probes minimizes their cleavage and consequently their fluorescent contribution Figure 5 3 illustrates results matches and mismatches between target and probe sequences in TaqMan PDARs for AD assays Livak et al 1995 Livak et al 1999 D Legend VIC Probe target sequence Probe target sequence raw mismatch higher T Allele X Quencher Allele J Y P mO irme m r Oz Polymerase Probe target sequence Probe target sequence R1558 mismatch higher T Figure 5 3 Target and Probe Sequence Interaction Table 5 1 shows the correlation between fluorescence signals and sequences present in the sample Table 5 1 Signal and Sequence Correlation A substantial increase in Indicates VIC dye fluorescence only
253. fluorogenic probe system Mol Cell Probes 13 5 329 340 1999 Hirayama Y et al Concentrations of thrombopoietin in bone marrow in normal subjects and in patients with idiopathic thrombocytopenic purpura aplastic anemia and essential thrombocythemia correlate with its mRNA expression of bone marrow stromal cells Blood 92 1998 46 52 Johnson M R Wang K Smith J B Heslin M J and Diasio R B Quantitation of dihydropyrimidine dehydrogenase expression by real time reverse transcription polymerase chain reaction Anal Biochem 2000 Feb 15 278 2 175 84 Krauter J M P Wattjes S Nagel O Heidenreich U Krug S Kafert D Bunjes L Bergmann A Ganser and G Heil 1999 Real time RT PCR for the detection and quantification of AML1 MTG8 fusion transcripts in t 8 21 positive AML patients Br J Haematol 107 80 85 Kruse N Pette M Toyka K and Rieckmann P Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood J mmunol Methods 210 2 195 203 1997 Lee L Y Patel S R Hackett N R Mack C A Polce D R El Sawy T Hachamovitch R Zanzonico P Sanborn T A Parikh M Isom O W Crystal R G and Rosengart T K Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121 Ann Thorac Surg 2000 Jan 69 1 14 23 discussion 23 4 Leutenegger C M C N Mislin B Sigrist M U Ehrengruber R
254. following table describes the electrical symbols that may be displayed on Symbols on Applied Biosystems instruments Instruments Symbol Description Symbol Description Indicates the On position of the Indicates a protective main power switch grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument Indicates the Off position of the main power switch a push push main power receive or supply alternating switch current or voltage Indicates a terminal that may Indicates a terminal that can be connected to the signal receive or supply alternating or ground reference of another direct current or voltage instrument This is not a protected ground terminal Ul D Indicates the On Off position of Indicates a terminal that can Safety Symbols The following table describes the safety symbols that may be displayed on Applied Biosystems instruments Each symbol may appear by itself or in combination with text that explains the relevant hazard see Safety Labels on Instruments on page xvi These safety symbols may also appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support documents Symbol Description Symbol Description Indicates the presence of a consult the manual for further laser inside the instrument and information and to proceed to proceed with appropriate with appropriat
255. for e Signal tightness and uniformity Do the raw spectra signals from replicate groups and controls exhibit similar spectral profiles If not the plate or sample block could be contaminated e Characteristic signal shape Do the samples peak at the expected wavelengths For example samples containing only FAM dye labeled TaqMan probes should not produce raw fluorescence in the wavelength of a VIC dye component A signal present in wells that do not contain the dye could indicate that the sample master mix or well contains contaminants e Characteristic signal growth As you drag the bar through the PCR cycles do you observe growth as expected Absent growth curves may indicate a pipetting error well lacks template e Signal Plateaus Do any of the signals plateau Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal Multicomponent The Multicomponent Plot displays a plot of normalized multicomponent data from a Plot single well of a real time run The plot displays the component dye signals that contribute to the composite signal for the well What to look for e Correct dyes displayed Does the plot display all dyes as expected The presence of an unexpected dye may be the result of an error in detector setup such as assigning the wrong reporter or quencher dye e ROX dye fluorescence level Does the ROX dye signal fluoresce below the repo
256. for the unknowns contained on a plate Statistical mean of the normally distributed quantity values for a replicate group wells with the same sample name detector and task Calculated as the sum of all quantity observations readings in the replicate group divided by the number of replicates Statistical standard deviation of the normally distributed quantity values for a replicate group wells with the same sample name detector and task The standard deviation is calculated using the nonbiased or n 1 method square root of sum of the squared normed residuals divided by n 1 degrees of freedom with n being the number of replicates The fluorescent dye or non fluorescent banned to the 3 end of the TaqMan Probe which reduces fluorescence of the reporter dye at the 5 end by FRET until the reporter dye is cleared during PCR The interval between two set points during which the instrument heats or cools the samples Unmodified spectral data detected between the wavelengths of 500 nm and 660 nm collected by the 7900HT instrument during a real time or end point run The physical device used to hold the PCR reactions during evaluation on the Applied Biosystems 7900HT Fast Real Time PCR System Examples of reaction formats include ABI PRISM 384 and 96 Well Optical Reaction Plates or the TaqMan Low Density Array Refers to real time PCR processing of individual plates by the 7900HT instrument for absolute and relative
257. from the following post analysis options see page 6 52 Reanalyze the run data Adjust the display settings for the results table plate grid and plate document plots Print elements of the plate document Export the plate document results table or plots Save the analysis Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 9 Chapter 6 Analyzing Real Time Data Opening the Run Data User Access There is no access requirement All users can open absolute quantification data that Requirement has been saved to the SDS Enterprise Database Opena Opening a Plate Document File Plate Document 1 2 3 Click or select File gt Open In the Look in field navigate to and select the plate document file Click on The SDS software displays the plate document file Configure the Analysis Settings for each marker as explained in Configuring the Analysis Settings on page 6 11 Opening a Plate Document from the SDS Enterprise Database 5 6 10 11 Click or select File gt Open Document from Database In the Plate Query dialog box configure the Find Plates Matching These Criteria table with parameters that correspond to the document of interest Click Search Find Plates Matching These Criteria a Contains 334 e Configure 2 Runtime Between PETO 12 13 PM as 12 13 PM queries here i Search Clear Row Clear All Appen
258. ftware does not remove it Note The outlier removal feature is optional and can be activated from the Analysis Settings dialog box see page 6 29 Outliers can also be identified and eliminated manually as explained on page 6 49 In addition to the Grubbs tests the algorithm used by the SDS software features additional rules for dealing with SDS data Unless the majority of samples in a replicate population are at maximum cycle the algorithm ignores max cycle wells wells with Cys equal to or greater than the Maximum cycle value Regarding The experimental reproducibility of Cy values for relative quantification assays Amplification decreases as the starting copy number approaches extremely low levels less than Beyond 100 copies Therefore PCR targets with low starting copy number and subsequent Reproducible high Cy values may have significantly diminished statistical reproducibility To avoid Limits Problems with reproducibility for low expression targets the limits of reproducibility must be determined experimentally for each relative quantification assay all targets and the endogenous control Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 19 Chapter 6 Analyzing Real Time Data Essential Experimental Components Endogenous Control Calibrator Sample Replicate Wells All relative quantification experiments require data from a second probe and primer set that amplifies an endogen
259. gents and samples of known quantities See page 3 15 for instructions NTC all detectors of negative control wells that contain PCR reagents but lack template Relative Target all detectors of wells that contain PCR reagents for Quantification the amplification of target sequences Endogenous g all detectors of wells that contain reagents for the Control amplification of the endogenous control sequence 2 Inthe well inspector click the field in the Task column for each detector entry and select the appropriate task from the drop down list The SDS software labels all selected wells with the task Selected wells m Unknown Task drop down list task applied ER A to selection 3 14 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Step 3 Configuring the Plate Document with Tasks 3 Repeat steps through 2 to apply any remaining tasks to the plate document 4 Choose from the following options If constructing a Then plate document for ai absolute quantification assign quantities to the standard wells of the plate document as explained below e allelic discrimination e f necessary set the passive reference for the e relative quantification plate document as explained on page 3 16 e dissociation curve analysis Otherwise program the method for your run as explained on page 3 17 Assigning For the SDS software to create a standard cu
260. gn to the marker The software highlights the selected detector IMPORTANT You cannot assign more than two detectors to a marker and the detectors cannot use the same reporter dye Detectors assigned to CYP 2C9 2 nares l JEW 2010 ViC Nan Flo l 6 Ifevaluating multiple loci repeat steps 3 through 5 to create additional markers as needed IMPORTANT You must configure a marker with two detectors before you can apply it to a plate document Note Click 7 for information on the features of the Marker Manager dialog box or to delete markers from the Markers list 7 Apply the marker s to the allelic discrimination plate document as explained in Copying and Applying Markers on page 3 13 3 12 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Step 2 Applying Detectors and Markers Copying and IMPORTANT Once you copy a marker to the plate document it is no longer linked to Applying Markers the corresponding entry in the Marker Manager Consequently if you modify a marker using the Marker Manager after you have copied it to a plate document you must remove the marker and copy it again to update the plate document with the changes 1 In the Marker Manager dialog box of the SDS software copy the allelic discrimination marker to the plate document a While pressing and holding Ctrl select the marker s you want to apply to the plate document b Click _ Sey ToPlate Docu
261. h expression level of rRNA even gross DNA contamination has a negligible effect on the relative quantification values obtained from the Low Density Array Single Plex The Low Density Array is recommended for use with single plex TaqMan reagents Reactions for example FAM dyes An external endogenous control assay for example FAM 18S or FAM GAPDH is used as a calibrator in relative quantification calculations Note For more information on relative quantification see ABI PRISM 7700 Sequence Detection System User Bulletin 2 Relative Quantitation of Gene Expression PN 4303859 Quality Control Functional verification of the preloaded probes and primers inside the Low Density Array is performed as part of the Applied Biosystems manufacturing quality control process Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 11 Chapter 4 Operating the Instrument About the Centrifuge System After the fill reservoirs of a Low Density Array have been loaded with the cDNA sample or control the centrifuge system is required to distribute the cDNA sample or control to the reaction wells The centrifuge system supports the simultaneous loading of up to 12 Low Density Arrays Required To properly centrifuge the Low Density Arrays you will need the following Centrifuge and centrifuge and centrifuge accessories Accessories e Sorvall Legend T Centrifuge Figure 4 7 Sorvall Legend T Centrifug
262. h selected detector relative to the expression level in the calibrator sample shown in the Calibrator drop down list Note Because the calibrator is compared to itself the expression level for the calibrator always appears as 1 1E 00 Table 6 1 on page 6 32 summarizes the circumstances in which the SDS software displays gene expression levels for sample replicate groups The shaded rows of the table indicate the combinations of data for which the software cannot calculate accurate expression levels Note The expression minimum 7 and maximum J symbols are described in detail on the page 6 33 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 31 Chapter 6 Analyzing Real Time Data Table 6 1 Summary of Expression Levels eeir Sample AC s Then expression Example for a 40 cycle experiment Calibrator Test EVERIS Calib AC Test AC Result Target ae a Displayed 25 32 RQ is displayed Endo lt 2 33 35 Bar is displayed Target Max lt A minimum gt 34 RQ is displayed Endo lt lt bar displays 7 35 30 Bar displays T Target lt Max A maximum 33 RQ is displayed Endo lt lt bar displays J 33 33 Bar displays 1 Target Max Max Undetermined RQ value is not Endo Max Max not displayed calculate Bar is not displayed Target lt Max Undetermined 30 Endo Max Max not displayed Target Max lt
263. h those of the other wells Contaminated Any material contaminating the sample block can affect the results For example Sample Block mineral oil reduces thermal transfer Residue from writing on reaction plates darkens the wells absorbing light The sample blocks should be periodically inspected for cleanliness Sample block contamination can be visualized by running a background plate and inspecting the resulting background signal for aberrant peaks above 2500 FSU see page 7 16 See page 7 14 for instructions on decontaminating the sample block Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 8 7 Chapter 8 Troubleshooting Improper or Damaged Plastics Low Copy Templates Use of Non Applied Biosystems PCR Reagents Only Applied Biosystems optical plates optical adhesive covers and optical flat caps should be used with the Applied Biosystems 7900HT Fast Real Time PCR System The plastics that comprise the optical parts undergo special testing for the absence of fluorescent impurities Optical plates are frosted to improve the degree and precision of light reflection Bent creased or damaged plastics may adversely affect the transmission of fluorescent signal or prevent proper sealing of a well resulting in evaporation change in sample volume and altered PCR chemistry Make sure to use the correct plastics and visually inspect each reaction plate before use Note See Appendix C
264. he instrument may pause up to 15 min to heat the heated cover to the appropriate temperature 6 When the run is complete a Analyze the run data as explained on page 6 12 b Set the baseline and threshold values for the analyzed data as explained on page 6 46 c Verify the performance of the 7900HT instrument as explained below Analyzing Data from RNase P Runs The install specification of the Applied Biosystems 7900HT Fast Real Time PCR System demonstrates the ability to distinguish between 5 000 and 10 000 genome equivalents with a 99 7 confidence level for a subsequent sample run in a single well The following equation verifies the 7900HT instrument install specifications CopyUnk 3 Scopyunk1 gt CopyUnk 3 Scopyunk2 where e CopyUnk is the Average Copy Number of Unknown 1 10 000 replicate population Ocopyunki 1S the Standard Deviation of Unknown 1 10 000 replicate population e CopyUnk is the Average Copy Number of Unknown 2 5000 replicate population OcopyUnk2 S the Standard Deviation of Unknown 2 5000 replicate population Note The values above can be obtained from the experimental report window Note Up to six wells from each replicate group in a 96 well TaqMan RNase P Instrument Verification Plate both standard 96 well and Fast 96 well plates can be ignored to meet specifications Note Up to 10 wells from each replicate group in a 384 well TaqMan RNase P Instrument Verific
265. hen to Perform Materials Required Changing the Plate Adapter Remove and replace the 7900HT instrument plate adapter after changing the sample block module format for example replacing a Standard 384 Well Block with a Fast 96 Well Block Note The sample block must be used with the corresponding plate adapter of the same plate format 2 3 32 inch hex key One of the following 384 Well Plate Adapter 96 Well Plate Adapter Fast 96 Well Plate Adapter TaqMan Low Density Array Adapter Ifthe instrument tray is inside the 7900HT instrument move the instrument tray to the OUT position a Start the SDS software b Click G or select File gt New c In the New Document dialog box click d In the new plate document select the Instrument tab e In the Real Time tab of the Instrument tab click open close The instrument tray rotates to the OUT position f Select File gt Exit The SDS software exits Remove the four screws attaching the plate holder to the plate arm Unscrew Unscrew Remove the plate adapter from the instrument tray Note If changing sample block formats for example replacing a Standard 384 Well Block with a Fast 96 Well Block store the plate adapter with the sample block module of the same format 7 12 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Changing the Plate Adapter 4 Place the new plate adapter
266. homozygosity for Allele X FAM dye fluorescence only homozygosity for Allele Y 5 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Overview Table 5 1 Signal and Sequence Correlation A substantial increase in Indicates both fluorescent signals heterozygosity Algorithmic The SDS software can analyze raw data immediately upon completion of an allelic Manipulation of discrimination run The term raw data refers to the spectral data between 500 nm to Raw Allelic 660 nm collected by the SDS software during the plate read During the analysis the Discrimination software employs several mathematical algorithms to generate from the raw data a more Data direct measure of the relationship between the spectra changes in the unknown samples The first mathematical algorithm involves the conversion of the raw data expressed in terms of Fluorescent Signal vs Wavelength to pure dye components using the extracted pure dye standards After the software identifies the dye components it determines the contribution of each dye in the raw data using the multicomponent algorithm About the After normalization the software processes all samples associated with markers that are AutoCalling configured for AutoCalling using the Applied Biosystems proprietary Maximum System Likelihood Algorithm The algorithm conducts a cluster analysis of the data based on the ratio of norm
267. hreshold Thus Xp X X 1 E y Ky where Xr threshold number of target molecules Cry threshold cycle for target amplification Ky constant D 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Comparative C Method of Relative Quantification A similar equation for the endogenous reference reaction 1s C Ry R x 1 Ep Kp where R threshold number of reference molecules R initial number of reference molecules ER efficiency of reference amplification Ct R threshold cycle for reference amplification Ka constant Dividing X by Ry gives the following expression X X x 1 E yx K T o X X T R ASAR AE eC R R CT R T R x l Ep The exact values of X and Ry depend on a number of factors including e Reporter dye used in the probe e Sequence context effects on the fluorescence properties of the probe e Efficiency of probe cleavage e Purity of the probe e Setting of the fluorescence threshold Therefore the constant K does not have to be equal to one Assuming efficiencies of the target and the reference are equivalent Ey Ep E X Cr y C 2x 1 E K R O or AC Xy X 1 E K where Xn X R the normalized amount of target AC Cx Cr p the difference in threshold cycles for target and reference Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide D 9 Appendix D Theory of Operat
268. ial In this configuration Figure 1 13 on page 1 19 the 7900HT instrument has the storage capacity and analysis capability to support medium to large scale gene expression and genotyping studies Moreover the SDS Enterprise Database functions as a repository for all SDS related data produced by the networked client computers used for data collection and analysis 1 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Managing Sequence Detection System Data Applied Biosystems 7900HT Data Collection Client Fast Real Time PCR System Desktop Computer Automation Controller Software Instrument Firmware Plate documents run from the database Thermal Cycling and Sequence Detection Serial Raw and Analyzed Data Data Collection Cable Saved to the Database Ethernet Database Server SDS Enterprise o Plate Analysis Documents Sessions Studies Ethernet Analysis Client Desktop Computer Instrument Firmware Plate Document Plate Documents Opened and Analyzed Created RQ Manager Software Hard drive s Analysis Sessions Analyzed as Studies SNP Manager Software Assay Plate Setup Files Analysis Sessions Analyzed as Studies Figure 1 13 Automated Operation of the of the 7900HT Instrument with SDS Enterprise D
269. ialog box or to Documents from view the procedures for importing or editing Plate IDs click 7 and see the Sequence aTemplate Detection Systems Software Online Help 1 Inthe SDS software open a plate document template from the Computer Hard Drive a Click gl or select File gt Open b Select File of type gt SDS 7900HT Template Document sdt c In the Look in field navigate to and select the plate document template d Click open e Go to step 2 SDS Enterprise Database a Select File gt Open Template from Database b In the Template Names field of the Select Template dialog box navigate to and select the plate document template c Click d Click Doe e Save the plate document template to the computer hard drive as explained on page 3 23 f Go to step 2 The SDS software displays the plate document template 2 Send the plate document template to the queue a Select the Instrument tab b Select the Queue tab 3 A Click Send To Queue 4 36 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Operating the Software without an SDS Enterprise Database 4 Configure the Template Batch dialog box with Plate IDs a Click new b In the Plate ID field of the New Plates dialog box and scan the bar code of the first plate in the batch using the hand held bar code scanner Am LASER HAZARD Exposure to direct or reflected laser light can burn the
270. ically remove the data of outlying replicate wells using the outlier removal algorithm See Automatic Outlier Removal on page 6 19 for an explanation of the algorithm e Omit replicates whose AC lt Instructs the software to remove from the analysis the data of any sample well whose AC is less than the number of cycles specified in the associated field This option is only available for multiplex experiments where similar target and endogenous control Cys may indicate that competition of the assays 1s affecting the amplification Note You can also identify and eliminate outliers manually as explained in Eliminating Outlying Amplification on page 6 49 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 27 Chapter 6 Analyzing Real Time Data Gene Expression Settings for Study 6 28 Settings PI Endogenous Control Detector The settings in this group box determine how the software handles the endogenous control data and RQ Min Max confidence values for your study These settings are crucial to the analysis and are set once for the study 1 m Calibrator Sample drop down list Calibrator Sample Sample 1 Endogenous Control Detector drop down list Control Type Multiplexed f Non tultiplexed Control Type option buttons RO Min Max Confidence Export Individual Acts RQ Min Max Confidence drop down list Fig
271. ick In the Saved Document dialog box click 4 Run the plate document and associated plate as explained on page 4 25 4 24 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Running a Single Plate Using the SDS Software Running a Single Plate Using the SDS Software User Access There is no access requirement All users can run plates that have been saved to the Requirement SDS Enterprise Database Running the Plate 1 Inthe SDS software select the Instrument tab of the plate document 2 Inthe Real Time or Plate Read tab of the Instrument tab click ren Close The instrument tray rotates to the OUT position 3 Place the prepared optical plate or Low Density Array into the instrument tray as shown below Before loading the plate or Low Density Array onto the instrument tray make sure that e The associated plate document is open in the SDS software The optical plate or Low Density Array has been sealed Well A1 zogo ogo zx RR I e goers ogo 823288 oe Notched corner Bar code Well A1 Notched corner Bar code IMPORTANT The A1 position is located in the top left side of the instrument Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 25 Chapter 4 Operating the Instrument 4 Do one of the following e If performing a real time run click start e If performing an end point
272. ick the plot or grid and select Save Plot Grid to Image File from the contextual menu Note Ifa pane cannot be exported as a graphic the contextual menu will not contain the Save Plot Grid to Image File option In the Save As dialog box navigate to the directory you want to receive the exported graphic file In the File name field and enter a name for the new file Click Save The software saves the plot or grid as a JPEG graphic in the designated directory Exporting Plate Document Data Exporting Data The SDS software can export raw or analyzed data in tab delimited txt format for from a Plate all or a select group of wells on a plate document The exported files are compatible Document with most spreadsheet applications To export an element of a plate document l Click the plate document to select it IMPORTANT The plate document must be the top most object in the workspace Click or select File gt Export Navigate to the directory you would like to receive the exported file s Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Exporting Plate Document Data 4 In the Export drop down list select the type of data you would like to export e Background Spectra The exported file contains the fluorescence readings for each well from the background component used to analyze the run e Clipped The exported file contains the average R and AR of the
273. icz 1983 During PCR if the target of interest is present the probe specifically anneals between the forward and reverse primer sites The 5 3 nucleolytic activity of the AmpliTaq Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target The probe fragments are then displaced from the target and polymerization of the strand continues The 3 end of the probe is blocked to prevent extension of the probe during PCR This process occurs in every cycle and does not interfere with the exponential accumulation of product The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR Because of these requirements any nonspecific amplification is not detected D 2 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Fluorescent Based Chemistries Basics of SYBR The SYBR Green 1 Double Stranded Binding Dye is used for the fluorescent Green Dye detection of double stranded DNA dsDNA generated during PCR The SYBR Chemistry Green Dye binds non specifically to dsDNA and generates an excitation emission profile similar to that of the FAM reporter dye When used in combination with a passive reference the SYBR Green 1 Dye can be employed to perform several SDS related experiments including quantitative PCR and dissociation cure analysis Figure D 2 illustrates the actio
274. ied Biosystems 7900HT Fast Real Time PCR System Site Preparation Guide PN 4317595 e Applied Biosystems 7900HT Fast Real Time PCR System Quick Starts for Allelic Discrimination PN 4351673 Absolute Quantification PN 4351672 Relative Quantification PN 4351674 e Sequence Detection Systems Software Online Help e Microsoft Windows Operating System Online Help e SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide PN 4351669 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 3 2 4 Chapter 2 Getting Started Powering On the 7900HT Instrument Powering On the Instrument Status lights The activation of the Applied Biosystems 7900HT Fast Real Time PCR System is sequential You must activate each component in a specific order for the system to initialize properly If performed out of sequence the components may not be able to establish the necessary communication connections required for operation Computer power button 7900HT instrument power button Monitor power button Zymark Twister Microplate Handler power button in rear Figure 2 1 Power Buttons of the Applied Biosystems 7900HT Fast Real Time PCR System IMPORTANT Power on the power to the instrument and the Plate Handler at least 10 min before use When activated the instrument heats the sample block cover to 105
275. ier Removal for Study Settings on page 6 27 for more information 7 Inthe Calibrator Sample drop down list select the sample that you want to use as the calibrator for your study Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 29 Chapter6 Analyzing Real Time Data 8 In the Endogenous Control Detector drop down list select the detector 10 11 12 representing the endogenous control for your study IMPORTANT The detector used as the endogenous control must be present in all sessions attached to the study Select the option button Multiplexed or Non Multiplexed to indicate the type of endogenous control used Note The Multiplexed or Non Multiplexed option buttons are active only 1f the sessions loaded for analysis contain both multiplex and non multiplex reactions In the RQ Min Max Confidence drop down list select the confidence value you want the software to use when generating error bars for the gene expression plot If desired select the Export Individual AC option button to instruct the SDS software to display the AC values for individual samples belonging to replicate groups If the option is not selected the software displays the average AC for the replicate group Click to accept the analysis settings and exit 13 Analyze the data as explained in Analyzing the Data on page 6 30 Analyzing the Study Data Analyzing the Data During the
276. ies with standard UL 3101 1 Safety Requirements for Electrical Equipment for Laboratory Use Part 1 General Requirements This instrument has been tested to and complies with standard CSA 1010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements This instrument has been tested to and complies with ICES 001 Issue 3 Industrial Scientific and Medical Radio Frequency Generators Safety This instrument meets European requirements for safety Low Voltage Directive 73 23 EEC This instrument has been tested to and complies with standards EN 61010 1 2001 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements and EN 61010 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials EMC This instrument meets European requirements for emission and immunity EMC Directive 89 336 EEC This instrument has been tested to and complies with standard EN 61326 Group 1 Class B Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements XXill Safety and EMC Compliance Information Australian EMC This instrument has been tested to and complies with standard AS NZS 2064 Standards Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radio frequency Equipment
277. if applicable and averages the resulting data Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 33 Chapter 6 Analyzing Real Time Data Viewing Relative The software displays the results of the relative quantification calculation in the Quantities in the Results table of the plate document Figure 6 13 shows an example of the results Analysis Table table containing the data from a TaqMan Human Cytokine Plate I _Postion Sample Demol ca Sample 1 ik Target _ 31 996471 Demote Sample 1 K Target Undetermined ai a thy 8 E F L Ree ie j E H fii les i Ei j J aa avg sa aco 5 177271 6 989E 1 0 ATSE 1 6 76172 S 1TT271 6 989E 1 0 1s A79E 1 6 76172 Baseline failed Rejected Figure 6 13 Analysis Table of the Absolute Quantification Plate Document The columns of the table are Position Displays the plate the sample is from and the position on the plate well number Sample Displays the name assigned to the well sample when the associated plate was run Detector Displays the name of the detector assigned to the sample well position Task Displays the task applied to the detector assigned to the sample well position C Displays the average calculated threshold cycle for the replicate group associated with the test sample Note The software displays Undetermined in the C column when the associated sample fails to produce
278. ification Overview Visualizing Outliers For any PCR experimental error may cause some wells to amplify insufficiently or not at all These wells typically produce Cy values that differ significantly from the average for the associated replicate wells If included in the absolute or relative quantification calculations these outliers can potentially result in erroneous measurements To ensure precise relative quantification replicate groups must be carefully examined for outlying wells The C vs Well Position view of the Amplification plot allows you to examine each set of replicate wells for outliers l 2 Select the Results tab If analyzing a Relative Quantification study select a plate in the Plates field and deselect all wells in the plate grid Repeat for each plate in the Plates field When you are finished the Amplification plot should not display any data If eliminating outliers from a Absolute Quantification Plate Document Select all wells of the plate by clicking the upper left corner of the plate grid Relative Quantification Multiple Plate Document a Select a plate in the Plates field to view b Select all wells of the plate by clicking the upper left corner of the plate grid In the Plot drop down list select Ct vs Well Position Inthe Well versus Threshold Cycle Amplification plot verify the uniformity of each replicate population by comparing the groupings of C values for the wells that
279. il ace Bowe a ae Rati Sate aaral kind rN aad Getaway bt Ble 6 23 Analyzing Ine Sway 22 dea iiare eats bees eee e eee Eee eee ke 6 26 About the Analysis Settings 0 0 00 cece eee eens 6 26 Specifying the Analysis Settings 2 0 00 ees 6 29 Analyzing the Study Data sous waive ea ut eho awe hee eee UNS eee 6 30 Viewing RESURS sanae sroine an ies tele ae ay by eh sa de Ges el wea an ee EO Mtn ak een Wd 6 31 Aerie ANANS S ica ke toe tone ate at ok Cen ee a tee ht ee eso ere ee 6 35 Section 6 3 Dissociation Curve Analysis 000 eee ee 6 37 OVENICW aser eooo t Ee eae es GoD Bees ae Oe ee es Bee ee 6 38 BeTOre VOU BEGIM 1 Sez 5 satis oh ee hed nee ds oe os we eh os ce ae be eee ae 6 39 AnalvSiS CHeCKIS grn oe minh waa eee a Melee e es eae bet ae 6 40 Opening Ihe RUN Data a2 4 duns saa aes aoa Seen ew ol Pa i Be ae ate Bl ae 6 41 Analyzing Ine RUN Wala per edb ea eee ee ee ee Ga oe at A 6 41 Determining T Values for the Analyzed Run 0000 eee eee 6 42 Afren TMS SPAIVANY SIS sess rads reinen orks apt a a gcd Aas ab pactostenea desk aes ad nel hy Dore ad ts ele ae cere 6 44 Section 6 4 Procedure Reference 0 000 cece ee 6 45 Setting the Baseline and Threshold Values 000 ccc eee eee 6 46 Eliminating Outlying Amplification 0 0 0 00 ee eee 6 49 ARE TING AIAIY SIS erreira ee ae dete nae ee ae Saat ae ah ey su Bek ac Acar eel a a Ee 6 52 Applied Biosystems 7900HT Fast Real Time PCR Sy
280. in the order that they appear in this document A 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 File Version Description Format Example Setup Table Files This line defines the version of SDS Assay Plate File format used to generate the document Note Assay Plate Setup Files of version 4 and higher cannot be imported directly into SDS 2 0 or SDS 2 1 SDS Setup File Version lt tab gt version number lt cr gt k SDS Setup File Version 3 Plate Characteristics 2 Plate Size Description Format Example 3 Plate ID Description Format Example This line defines the number of wells in the plate modeled by the file for example 384 or 96 Output Plate Size lt tab gt number of wells lt cr gt Output Plate Size 384 This line defines the ID of the Assay Plate Normally this is a bar code that is printed on the plate Output Plate ID lt tab gt plate id lt cr gt Output Plate ID 384N75822034 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 5 Appendix A Software Reference Detector Definitions Element numbers 4 to 6 define the detectors that appear in the well descriptions that follow in a later section The detector definition consists of three sections the declaration of the number of detectors the detector list header and the detector list 4 Number of Detec
281. ing the Plate Document Method on page 3 17 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 15 Chapter 3 Preparing a Run Step 4 Setting the Passive Reference and Omitting Wells User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to change the passive reference or omit wells from use Setting fusing an Applied Biosystems chemistry use the default Passive Reference setting the Passive Applied Biosystems chemistries use the ROX passive reference dye molecule If Reference running a custom chemistry select a dye to use as a passive reference for the run Note Applied Biosystems recommends using a passive reference to normalize the signals from the reporter dyes 1 Inthe Passive Reference drop down list select the appropriate reference dye Passive Reference drop down list Select the appropriate passive reference 2 Ifnecessary omit wells from use as explained below Otherwise program the method for the run as explained on page 3 17 Omitting Wells The SDS software allows you to omit wells from use before or after running a plate from Use IMPORTANT If you remove a well removed from use before you run a plate document the SDS software will not collect data for the well during the run However if you remove a well from use after you run a plate document the software excludes
282. ing the SDS sofiwarS 2 444 oe hed eoe es o Shoo noha es Rass 7 55 Note The SDS software is a multicomponent system that must be maintained to ensure optimal operation of the Applied Biosystems 7900HT Fast Real Time PCR System Although an Applied Biosystems service engineer will complete most of the maintenance this section discusses important issues that you should understand Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 53 Chapter 7 Maintaining the Instrument General Computer Maintenance Maintenance The computer connected to the 7900HT instrument requires regular maintenance to Schedule ensure reliable operation of the Applied Biosystems 7900HT Fast Real Time PCR System components Applied Biosystems recommends the following tasks as part of routine maintenance of the computer system Table 7 4 Maintenance Schedule for the Instrument Computer Maintenance Task Perform Archive or Remove Old SDS Files Weekly Defragmenting the Hard Drive Monthly or before fragmentation reaches 10 Upgrading the Operating System Software When available advisable Upgrading the SDS Software When available Developing a Data Management Strategy Applied Biosystems recommends developing a strategy for dealing with the files produced by the SDS software During a single day of real time operation the Applied Biosystems 7900HT Fast Real Time PCR System can generate over 200 MB of dat
283. ing the success or failure of the signature Click x to continue You are attempting to change the For Property X of the currant plat document Description of the action that requires a signature Enter your user name Enter your password Figure 4 2 Electronic Signature Verification Dialog Box Options Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Before You Begin Before You Begin Pre Run The following tasks must be complete to run plates on the 7900HT instrument See Checklist the associated page number for details on each procedure Done Check See Page A background run has been performed in the last month 7 16 A pure dye run has been performed in the last 6 months 7 20 The instrument tray does not contain a plate 4 44 IMPORTANT The instrument tray must be empty to begin a run If the instrument tray contains a plate you must eject and remove it If using an Automation Accessory also check the following The output stack does not contain plates Workflow Overview 1 Prepare your experiments for use on the 7900HT instrument Prepare optical plate s See page 4 8 Prepare TaqMan Low Density Array s See page 4 10 2 Prepare and run the 7900HT instrument Stand alone Operation see page 4 23 Automated Operation using the SDS Enterprise Database see page 4 32 Automated Oper
284. ining the file or files of interest While pressing and holding the Ctrl key select the plate document file s to add to the plate queue The software highlights selected files IMPORTANT A plate document must contain a bar code before you can add it to the plate queue See page 3 25 for more information on configuring a plate document with bar code information Click Open The Automation Controller Software adds the plate document s to the Plate Queue Note After you have added a plate document to the plate queue the software locks the file preventing any changes from being made to it until the plate document has been run or removed from the queue Removing Plate To remove plate documents from the plate queue Documents from the Plate Queue To remove Then specific plate 1 While pressing and holding the Ctrl key select the plate documents from the document files to remove plate queue The software highlights selected files 2 Select File gt Clear Selected Plate s 3 Click _ te The software removes the selected plate documents from the plate queue all plate documents 1 In the Automation Controller Software select File gt from the plate queue Clear All Plates 2 Click _ tes The software removes all plate documents from the plate queue 4 40 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Running Plates Using the Automati
285. into the sealer so that the sealer s carriage stylus assembly travels toward the fill reservoir end of the Low Density Array Always seal in the direction indicated by the arrows etched into the base of the sealer to ensure that the cDNA sample or control is pushed from the Low Density Array s channels into its fill reservoirs Positioning the The sealer is best operated by pushing the carriage away from you in a near to far Sealer motion not in a horizontal side to side motion Follow the procedure below to correctly position the sealer l Inserting a Card 1 Into the Sealer Place the sealer on a sturdy lab bench approximately waist high so that it can be easily used Turn the sealer so that the front end where sealing starts is closest to you and the back end is farthest from you Note In the correct position the arrows on the sealer are pointing away from you Carriage Place the sealer s carriage in its starting position IMPORTANT Never insert a Low Density Array into the sealer if the carriage is not in its starting position If you move the carriage across the Low Density Array to return it to its starting position serious damage to the Low Density Array will occur Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 19 Chapter 4 Operating the Instrument 2 Insert a Low Density Array into the sealer a Orient the Low Density Array in the proper directi
286. ion Rearranging gives the following expression Cr A Xy Kx 1 E The final step is to divide the Xy for any sample q by the Xy for the calibrator cb AC ae 2 Kx 1 E L ian AC Neb KOFE where AAC ACr q ACr For amplicons designed and optimized according to Applied Biosystems guidelines amplicon size lt 150 bp the efficiency is close to 1 Therefore the amount of target normalized to an endogenous reference and relative to a calibrator is given by 5 AACr D 10 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Limited Warranty Statement Warranty Applera Corporation through its Applied Biosystems Group Statement Applied Biosystems warrants to the customer that for a period ending on the earlier of one year from the completion of installation or fifteen 15 months from the date of shipment to the customer the Warranty Period the Applied Biosystems 7900HT Fast Real Time PCR System purchased by the customer the Instrument will be free from defects in material and workmanship and will perform in accordance with the installation specifications set forth in the system specifications sheet which accompanies the instrument or which 1s otherwise available from an Applied Biosystems sales representative During the Warranty Period if the Instrument s hardware becomes damaged or contaminated or if the Instrument otherwise fails to meet the S
287. ion The Condition operator establishes the relationship between the Column category Operators and the Value 1 and Value 2 criteria by defining the action of the evaluation The following list summarizes the possible conditional operators and their corresponding actions e gt Greater than Value 1 e gt Greater than or equal to Value 1 e lt Less than Value 1 e lt Less than or equal to Value 1 e equals Equal to Value 1 e lt gt Does not equal Value 1 e between Is greater than Value 1 but less than Value 2 e contains Contains the alphanumeric string found in Value 1 Value 1 and 2 The Value 1 and Value 2 criteria define the condition s for the evaluation Criteria Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 21 Appendix A Software Reference Connecting SDS Software to the Database Required Information Disabling Enabling the Database Functions in the SDS Software IMPORTANT Only users belonging to the Administrator or Service Engineer user group can modify the database parameters for an SDS client application You need the following information to connect a SDS client application to the database e Host Name The Host Name setting or the IP address for the SDS Enterprise Database Check with your database administrator for the correct identification notation for the server running the database on your network e Port Number The Port Number default setting is
288. is Side Out label IMPORTANT Use blank balance cards to fill any remaining positions in the racks Note The card holder supports the Low Density Array fill reservoirs during centrifugation 4 Place a filled card holder in the bucket so that the This Side Out label faces the front of the bucket which has the Sorvall emblem on it Sorvall emblem here 4 16 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Preparing TaqMan Low Density Arrays for Use Placing the 1 Make sure the centrifuge is powered on but is not rotating Buckets at the 2 Open the centrifuge cover by pressing the OPEN button Centrifuge OPEN cover button 3 Place a loaded bucket onto an open rotor arm of the centrifuge Note Make sure the bucket can swing easily within its slotted position on the rotor arm 4 Place the remaining buckets onto the rotor arms per step 3 N ozoli The manufacturer recommends running the centrifuge with all four buckets even if only two buckets contain Low Density Arrays N 7 1010 Make sure the buckets and their contents are balanced Opposing buckets should have matching weights If the buckets are not fully loaded with Low Density Arrays containing the sample specific PCR reaction mix place blank balance cards and card holders into the buckets 5 Close the centrifuge cover Centrifuging 1 Press the START but
289. is the negative of the rate of change in fluorescence as a function of temperature Figure 6 16 shows the plots accessible from the Plot drop down list Setup Instrument Results Dissociation Curve Setup Instrument Results Dissociation Curve l Dissociation Plot 3 Dissociation Plot H 5 000 E1 4 000 E1 3 000 amp 1 2 000 E1 1 000 amp 1 0 000 Temperature 7 Step Stage 6 step 1 z Raw Plot Derivative Plot Detector SYBR Plot EERI Step Stage 6 step 1 x Detector SYBR Plot eiiReEii Figure 6 16 Plots of the Dissociation Curve Tab e Detector drop down list Chooses the data displayed within the plot based on detector name Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 43 Chapter 6 Analyzing Real Time Data Determining 1 Move the pointer over the green T line located on the Y axis line of the plot Tm Values 2 Click and drag the T line to the maximum point of the derivative plot of interest The SDS software displays the T for the curve below the T line Setup Instrument Results Dissociation Curve Dissociation Plot 5 000 E1 4 000 E 1 3 000 E1 Derivative 2 000 E1 1 000 E 1 0 000 Tm display and slider Temperature Detector SYBR Plot BEEN BY Step Stage 6 step 1 Note The apex of the curvature of represents the maximum rate of change in normalized fluo
290. isplays the Connected icon Hi tometa Patene IN the status bar when a plate document is open See About the Message Bar on page 2 9 for more information About the All software operations and displayed information occur inside the workspace of the Software SDS software The workspace provides quick access to all elements of the software Interface through the menubar and a pair of toolbars Figure 2 3 summarizes the features of the user interface of the SDS software Menubar Window buttons Contains a directory of menus Minimize maximize oy governing the operation of the or close xJ the window software File Edit view Tools Instrument Analysis Window Help OR x A o Ee ane eo Am E Display Toolbar Contains icons for controlling the display of information in the SDS software workspace see page 2 8 General Toolbar Contains icons for controlling the basic functions of the software see page 2 8 Ready M ironed Message Bar Instrument Connection Icon Displays messages that indicate Indicates the status of the the status of the instrument and connection to the 7900HT the SDS software see page 2 9 instrument see page 2 9 Figure 2 3 Elements of the SDS Software User Interface Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 7 Chapter 2 Getting Started About the The general toolbar contains tools used to control the basic functions of the software
291. ither e Select an existing study or e Click _ new and configure the Create New Study dialog box then click OK Name Enter a name for the study up to 128 characters Creator Not editable Displays your user account name Description Enter a brief description of the study up to 255 characters 3 Click 4 Prepare and load the plates onto the Plate Handler as explained in Loading Plates onto the Plate Handler on page 4 41 IMPORTANT The Save Analysis Results check box in the Database Options dialog box must be selected for the Automation Controller Software to associate session data default is ON When the option is selected the software automatically performs a primary analysis of each plate document it runs and saves the results to the database as an analysis session To access the Save Analysis Results option by selecting Enterprise gt Database Options Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 33 Chapter 4 Operating the Instrument Operating the Software without an SDS Enterprise Database 4 34 Overview Workflow The first step in configuring the Applied Biosystems 7900HT Fast Real Time PCR System for automated operation is to add plate documents to the plate queue The plate queue is a list of plate document files that the Automation Controller Software uses to identify and run associated plates during automated operation By
292. ixed position bar code reader and position the scan head of the reader as far as possible from the plate while maintaining the orientation towards the bar code on the plate see below Scan head of the fixed position bar code reader Black positional adjustment knob While watching the Terminal dialog box slowly adjust the orientation of the fixed position bar code reader until the percent successful reading displays the highest number possible ive E1E 1 I FP Percent successful reads Note It may be helpful to briefly place a sheet of white paper in front of the plate bar code to view the area scanned by the laser When satisfied with the alignment tighten the black positional adjustment knob on the fixed position bar code reader Restore the fixed position bar code reader to normal operation a In the Edit Configuration dialog box change from Test back to Serial on Line psc LD11000 Edit Configuration ES ajajaje 2 a 2 Operating modes selection Test Device Configuration On line E tran Numofslaves Test Operatina mode options b In the Device Control dialog box confirm that EEPROM 1s still selected and click Send c In the New Decision dialog box click YES to save to EEPROM The bar code reader stops scanning the plate bar code and resumes normal operation Click Exit m to quit the LDHOST window The LDHOST window closes Mode drop down list
293. k My Computer gt AppliedBiosystems gt SDS2 2 1 Software from a 2 Create a copy of the SDS 2 bat file and rename it such as SDS_RESULTS bat Command Line 3 Using the Notepad application edit the contents of the batch file as follows Example Code A 17 SDS2 bat Batch File The text of the batch file appears as follows set classpath service config service Lib audit jar service Lib rtc jar service Lib ab regres jar service Lib translation jar service Lib apv jar service lib coreutil jar service JMS lib fmprtl zip service Lib coregui jar service lib EmlException jar service lib emlhandler jar service lib esig jar sf Service lib e sig exception ars services lib Coreutil jar set path 1lib algorithm bin win3s2 labs tpaths jre bin java Dab home C AppliedBiosystems SDS2 2 service Dcom apldbio sds global approot Dcom apldbio sds global config config config properties Xms110m Xmx300m Dsource batchfile Ddefport 8080 classpath classpath 1lib SDS2 jar lib common jar lib sdsdb jar lib ojdbcl14 jar lib accesscontrol jar lib sdsdb jar lib activation jar Lip Maid jar laib adccesscont rol jar lib Coreutil ar lib EmLException jar com apldbio sds shell SDSMain In the last line replace com apldbio sds shell SDSMain with one of the following to generate e Multicomponent data for an SDS plate document file enter com apldbio sds MulticompAnalyzerMain e Analyzed data for an SD
294. katsume P Rinaldo and K Narisawa 2000 Mutation detection by TaqMan allele specific amplification application to molecular diagnosis of glycogen storage disease type Ia and medium chain acyl CoA dehydrogenase deficiency Hum Mutat 2000 15 2 189 96 Germer S and Higuchi R Single tube genotyping without oligonucleotide probes Genome Res 9 1 72 78 1999 Happich D Schwaab R Hanfland P and Hoernschemeyer D Allelic discrimination of factor V Leiden using a 5 nuclease assay Thromb Haemost 82 4 1294 1296 1999 Holloway J W Beghe B Turner S Hinks L J Day I N and Howell W M Comparison of three methods for single nucleotide polymorphism typing for DNA bank studies sequence specific oligonucleotide probe hybridisation TaqMan liquid phase hybridisation and microplate array diagonal gel electrophoresis MADGE Hum Mutat 14 4 340 347 1999 Livak K J Allelic discrimination using fluorogenic probes and the 5 nuclease assay In Process Citation Genet Anal 14 5 6 143 149 1999 Mizugaki M M Hiratsuka Y Agatsuma Y Matsubara K Fujii S Kure and K Narisawa 2000 Rapid detection of CYP2C18 genotypes by real time fluorescence polymerase chain reaction J Pharm Pharmacol 2000 Feb 52 2 199 205 Paris PL Kupelian P A Hall J M Williams T L Levin H Klein E A Casey G and Witte J S Association between a CYP3A4 genetic variant and clinical presentation in African American
295. la manipulation et l limination des d chets WARNING Hot lamp AVERTISSEMENT Lampe br lante WARNING Hot Replace lamp with an Applied Biosystems lamp AVERTISSEMENT Composants br lants Remplacer la lampe par une lampe Applied Biosystems CAUTION Hot surface ATTENTION Surface br lante DANGER High voltage WARNING To reduce the chance of electrical shock do not remove covers that require tool access No user serviceable parts are inside Refer servicing to Applied Biosystems qualified service personnel DANGER Haute tension AVERTISSEMENT Pour viter les risques d lectrocution ne pas retirer les capots dont l ouverture n cessite l utilisation d outils L instrument ne contient aucune pi ce r parable par l utilisateur Toute intervention doit tre effectu e par le personnel de service qualifi de Applied Biosystems DANGER Class 3B laser radiation present when open and interlock defeated Avoid direct exposure to laser beam DANGER Class 3B rayonnement laser en cas d ouverture et d une neutralisation des dispositifs de s curit Eviter toute exposition directe avec le faisceau DANGER Class 3B laser radiation when open Avoid direct exposure to laser beam DANGER Class 3B rayonnement laser en cas d ouverture Eviter toute exposition directe avec le faisceau DANGER Class 3B laser radiation present when open and interlock defeated Do not stare directly into the beam
296. last three data points collected during the extension phase of each cycle repetition for each well e Dissociation Curve The exported file contains Temperature Data For each well in use on the plate the file displays the calculated temperature of the wells during each data collection reading of the temperature ramp Raw Data For each well in use on the plate the file displays the R of the well during each data collection reading of the temperature ramp Derivative Data For each well in use on the plate the file displays the first derivative data during each data collection reading of the temperature ramp e Multicomponent The exported file contains Amounts the calculated dye components in a single well throughout all stages of the PCR that were labeled with data collection icons Pure spectra component data Calculated inverse matrix Singular values of the inverse matrix e Pure Spectra The exported file contains the fluorescence readings for each well from the pure spectra calibration component used to analyze the run e Raw Spectra The exported file contains the unmodified fluorescence readings taken for each spectral bin during the course of the run e When exported the software creates a directory and saves each the raw spectra data for each well in a separate txt file e Results Table The exported file contains the contents of the table pane of an analyzed plate document N
297. late document Plate Document you must remove the detector from the plate document and copy it again to update the plate document with the changes 1 In the Detector Manager dialog box of the SDS software copy the detectors to the plate document a While pressing and holding the Ctrl key select the detectors you want to apply to the plate document The software highlights the selected detectors b Click Copy To Plate Document i The software adds the detectors to the well inspector of the plate document Plate document eh Fie ot Veew Tools buie Ane Parciow Heip E E Re Sw Fy Usiieg j Mente niet ification Copied detector Detector Manager Detector selected for copying 2 Click _ pone to close the Detector Manager 3 Inthe plate grid select the wells containing the assay for the first detector Note For easier selection of plate grid wells use the Ctrl and Shift keys to select wells individually or in groups See page 2 23 for more information 4 Apply detector to the selection by clicking the check box for the detector in the Use column of the well inspector 7 Wells B2 Sample Name 52 Joe es E bine bisse F hissa F Detector added to selected wells of the plate document Use check box fal Daer rhea Lirih asg 5 Repeat steps 3 through 4 to apply the remaining detectors to the plate grid 6 Configure the plate document with detector tasks a
298. latform for the Low Density Array and a track for the stylus assembly Figure 4 9 Sealer Components Shown with Cover Removed The microfluidic card sealer is designed to be placed on a standard laboratory bench It requires no electrical power Loading the TaqMan Low Density Arrays Equipment and Materials Needed Guidelines for Loading the Cards You need the following equipment and materials to load the sample specific PCR reaction mix into the fill reservoirs of the Low Density Arrays Rainin F 100 micropipette 100 uL Rainin Fine Point pipet tips 100 uL TaqMan Low Density Array s Sample specific PCR reaction mix see the protocol accompanying your Low Density Array chemistry for instructions on preparing the reaction mix Follow the guidelines below to ensure proper loading of the Low Density Arrays Do not remove a Low Density Array from its packaging until the packaging has reached room temperature and you are ready to load it with the sample specific PCR reaction mix IMPORTANT Prolonged exposure to indoor lighting can photo degrade the fluorescent probes contained inside the Low Density Array Do not expose the Low Density Array to sunlight Do not twist or bend the fill consumable portion of the Low Density Array Figure 4 5 on page 4 10 before loading IMPORTANT To successfully load the sample specific PCR reaction mix it is critical that the main channels leading from the Low Density Array s fill
299. lay Settings Printing a Report Exporting Plate Document Data If using the SDS Enterprise Database all users can perform the tasks explained below except for saving the results Only users belonging to the Scientist or Administrator User Group can save analyzed data to the database as a session Changing the Display Settings 0 0 cece eens see below PU a Report no ecb ecw t renra EEEE ce eee brates oR pees see below Exporting Plate Document Data 0 0 0 cece ee see below Savine Me Reilis gs hoe denne ee DOIT KE TEER EE EEE KERRIE EKE EERUN 6 53 Before printing or exporting the analyzed data you can reconfigure the appearance of several elements of the plate document including the results table plate grid and most plots 1 Click ml or select View gt Display Settings 2 Inthe Display Settings dialog box click 7 for further instructions on modifying the display settings The SDS software can print a report of the analyzed data containing individual or multiple elements of the plate document 1 Click amp or select File gt Print Report 2 In the Print Report dialog box click 7 for instructions on setting up previewing and printing the report Exporting Plate Document Data as a Tab Delimited Text File The SDS software can export raw or analyzed data in tab delimited txt format for all or a select group of wells on a plate document The exported files are compatible with most sp
300. lays the results in the Results tab 2 Choose from the following e fusing the AutoC algorithm to set the baseline and threshold values for the detectors on the plate verify that the software has called the settings correctly for each detector as described on page 6 46 e If you choose to set the baseline and threshold values manually set them for each detector on the plate as explained on page 6 46 3 If you choose to remove outliers manually visualize and eliminate outliers from the analyzed run data as explained on page 6 46 6 12 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Viewing Results Viewing Results Viewing the The software displays the standard curve generated from the run data within the Standard Curve Results tab of the plate document The standard curve plot displays the unknown samples on a graph of C threshold cycle vs Quantity LogN Figure 6 5 illustrates the components of the standard curve plot Setup Instrument Results Help button Standard Curve Plot Detector RNase P gt Detector drop down list Legend m Standards Legend box s Unknowns Hide Unknowns Hide Unknowns button Standard Cure Slope 3 3984165 Y Inter 37 091667 Standard Curve box R2 0 99807 143 Standard curve plot CeCe EEC PTT TT TTT TET TT ET EEE BRRGARRRARERERM RRR JA a ae a eh PEE E E E E E a a TY CS a CCCP EEE PTT TPE TTT TT TE TT ET ET ET T ER
301. le versus treated samples e A resting sample versus activated samples Note The SDS software combines the data from replicate calibrator wells at the aC level of the relative quantification calculation whether the replicates are present on a single or multiple plates For relative quantification studies Applied Biosystems recommends the use of three or more replicate reactions per sample to ensure statistical confidence Replicates allow you to e Preserve Data If an amplification fails in one well replicate wells can potentially provide data This point is especially true in the case of endogenous controls which upon failure may invalidate the results for the entire plate e Remove Outliers The use of replicate populations provide the opportunity for the visualization and removal of outliers e Ensure Statistical Reproducibility In general the use of replicates ensures a greater degree of experimental reproducibility by providing a means to identify and refute anomalous data caused by experimental error such as contamination pipetting errors and so on 6 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Getting Started Getting Started Using the SDS For specific instructions on any procedure described within this section refer to the Software Sequence Detection Systems Software Online Help To get help at any time during the Online Help procedure click 2 locat
302. lection 9 Seal the plate using an optical adhesive cover or optical flat caps 10 Create a plate document template for the custom Pure Dye plate as explained below Constructing a 1 Start the SDS software Custom Pure Dye Plate Document Template a Select Tools gt Dye Manager b In the Dye Manager dialog box click ne c In the Add Dye dialog box enter a name for the custom dye and click k The software adds the new dye to the Custom dye list Add the new dye to the software using the Dye Manager d Repeat steps b and c to add any additional custom dyes to the Dye Manager e Click Doe The SDS software makes the new dyes available to pure dye plate documents 3 Create a custom pure dye plate document for the run a Click or select File gt New b Configure the New Document dialog box with the following options Assay Select Pure Spectra Container Select the appropriate plate format Template Select Blank Template c Click The software creates a new plate document 7 28 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Adding Custom Dyes to the Pure Dye Set 4 Apply pure dyes to the custom plate document a Select the wells containing the custom dye b In the Dyes drop down list in the Setup tab select the appropriate dye The software applies the dye to the selected wells c Repeat steps a and b to configure th
303. led do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can cause painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons Moving and Nee Do not attempt to lift or move the computer or the monitor Lifting Stand without the assistance of others Depending on the weight of the computer and or the Alone Computers monitor moving them may require two or more people and Monitors Things to consider before lifting the computer and or the monitor e Make sure that you have a secure comfortable grip on the computer or the monitor when lifting e Make sure that the path from where the object is to where it is being moved is clear of obstructions e Do not lift an object and twist your torso at the same time e Keep your spine in a good neutral position while lifting with your legs e Participants should coordinate lift and move intentions with each other before lifting and carrying e Instead of lifting the object from the packing box carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box Operating the Ensure that anyone who operates the instrument has Instrument e Received instructions in both general safety practices for laboratories and specific safety practices for the instrument e Read and understoo
304. lid target and endogenous Down Arro control Cys Crs less than the maximum cycle and e A test sample yields an undetermined target C and a valid endogenous control At the levels above the test sample does not contain enough target cDNA for an accurate comparison with the calibrator sample Therefore the stated expression level is the maximum possible value for the associated test sample About Up Arrows The software displays an up arrow to indicate that a level of expression represents the minimum target gene expression level The software displays the up arrow on all Up Arrow associated sample bars when e The target sample for the detector group yields valid target and endogenous control Cys Crys less than the maximum cycle and e A calibrator sample yields an undetermined target C and a valid endogenous control At the levels above the calibrator sample does not contain enough target cDNA for an accurate comparison with the test sample Therefore the stated expression level is the minimum possible value for the sample About the The software displays error bars for each column in the plot if the associated Error Bars expression level was calculated from a group of two or more replicates The error bars display the calculated maximum RQMax and minimum RQMin expression levels that represent standard error of the mean expression level RQ value Collectively the upper and lower limits define the region of
305. llelex DONERE i Allele S E A Select Allele X le le le ey O T NTC Allele X homozygotes 1 0 2 0 3 0 Allele X CYP 2C19 2 2 5 Repeat steps 3 through 4 to apply calls to the rest of the samples within the plot Call Symbol Definition Allele X Homozygous for the allele displayed on the associated axis of the Allelic Discrimination Plot Allele Y Both Heterozygous Alleles X and Y NTC E No Template Control Undetermined xX Unknown Unlabeled Note You can adjust the appearance of the allelic discrimination plot or the data points it contains using the Display Settings dialog box See the Sequence Detection Systems Software Online Help for more information Allele Y homozygotes Allele X Allele Y heterozygotes Legend Allele X Allele Y Allele X amp Y E NTC Undetermined Allele Y CYP 2C19 2 1 Allele X homozygotes No amplification Allele X CYP 2C19 2 2 6 If evaluating for multiple markers do the following a In the Marker drop down list select a different marker b Repeat steps 2 through 5 for the new marker c Repeat steps a and b until the alleles for each marker have been called Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 5 15 Chapter 5 Analyzing End Point Data Scrutinizing the 1 Verify the calls for the NTC Allele X and Allele Y controls
306. lt See Page Hide lt pane or plot gt Hides the pane or view 2 21 Save lt pane or plot gt Opens the Export Graphic dialog box for exporting the A 16 to Image File selected view or pane as a JPEG graphic file Display Settings Opens the Display Settings dialog box which allows 3 22 you to modify the appearance of the view pane or plot Lesson 6 Using Keyboard Shortcuts Exercise Closing The SDS software provides keyboard shortcuts for invoking the major functions of the the Plate software A keyboard shortcut is a combination of two keys Ctrl and another key Document _ that when pressed in unison instruct the software to perform an action The software lists the keyboard shortcuts next to the options in each menu on the menu bar To demonstrate the use of keyboard shortcuts close the open plate document as follows 1 Simultaneously press the Ctrl and W keys Ctrl W 2 When prompted to save the plate document click Note The Sequence Detection Systems Software Online Help contains a complete list of the keyboard shortcuts for the SDS software To view the list open the online help as explained in Using the SDS Software Online Help on page 2 3 2 28 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Using SDS Plate Documents Using Multiple Plate Documents Elements of a Plate Document Plate Grid Using SDS Plate Documents Although the SDS softwar
307. ludes a copy of the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide which provides information for integrating the database into a laboratory workflow The guide includes e Detailed information about the SDS Enterprise Database API e Supporting information such as sample code javadoc reference and connectivity requirements for the database Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Getting Started In This Chapter Getting Started oa sks veaceehaneserbacdeddnrseeb ses aanceaencsseses 2 2 Powering On the 7900HT Instrument 0 0 0 0 0 0c eee 2 4 Usmo the SDS SOT a oho 4444 4 666 8G AE 2 7 Dasic Software Se Tutorial sses ow hee 4 bed eo bee cb Gb eee so enseieds 2 10 Cee SDS Plate DOCS sno osha 5 oe r re 69544 45 4 8 4 RS 2 29 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 1 Chapter 2 Getting Started Getting Started Before You Begin Procedure Quick Reference 2 2 If this is your first time using the Applied Biosystems 7900HT Fast Real Time PCR System consider completing the Basic Software Skills Tutorial on page 2 10 before continuing The tutorial will provide you with the fundamental knowledge required to operate the SDS software and will teach you timesaving techniques to allow you to use it quickly and efficiently The following table contains a list o
308. m Optical Adhesive Covers quantity 100 Optical Adhesive Covers quantity 25 ABI PRISM Optical Caps flat caps only Use these Not these ABI PRism 96 Well Compression Pads e ABI PRism Optical Reaction Standard pad for manual operation Optical Caps round caps e 96 Well Septa Optical 96 Well Fast Thermal Cycling Plates ABI PRISM Optical Adhesive Covers quantity 100 Optical Adhesive Covers quantity 25 TaqMan Low Density Arrays None self sealing None self sealing Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 7 Chapter 4 Operating the Instrument Preparing Optical Plates for Use Preparing Plates for Use on the 7900HT Instrument samples to the appropriate wells of an optical plate 1 Prepare the reactions in an optical plate by aliquoting reagents enzyme and IMPORTANT The arrangement of the reactions samples and assays on the plate must match the configuration of information sample names and detectors markers on the corresponding plate document Figures 4 3 and 4 4 on page 4 9 Seal the optical plate with a seal that is compatible with the 7900HT instrument Note See Compatible Consumables on page 4 6 for a list of seals for use with the Applied Biosystems 7900HT Fast Real Time PCR System to eliminate any air bubbles that may be present Briefly centrifuge the pla
309. me added to the C pplied Biosystems SDS Docume study File Search Search in laplied Bi Include Subdirectories Show Objects Matching This Criteria o Logic Column Condition J Axalue IF Mare contains rr S r i Query designed to find plate documents with file Search Clear D Append Results names containing RQ Search Results Existing Plates In The Study Plate documents MEM pplied Bios 5 Document Ra found by the software 2 8 E ATIE Wns SDS Documents Ra Clear All Humber of Plates Study can hold 10 6 free OK Cancel Note See Using the Search Tool on page A 20 for more information on searching for plate documents 3 Repeat step 2 to add additional files to the study up to 10 plates Note You can remove plates from the study by selecting the file from the File list field and then clicking Remove 4 When you finish adding files to the study click 5 Configure the analysis settings for the study as explained in Configuring the Analysis Settings on page 6 26 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 25 Chapter 6 Analyzing Real Time Data Analyzing the Study User Access Requirement Configuring the Analysis Settings There is no access requirement All users can analyze absolute quantification data that has been saved to the SDS Enterprise Database Before
310. ment to copy the markers to the plate document c In the Copy Markers To Plate dialog box click ok Copy Markers To Plate Ea The Following markers have been copied to the plate CYP cigo Markers copied to CYP 2c9 2 the plate document Ok d Repeat steps a and c to copy additional markers to the allelic discrimination plate document as needed 2 Click _ bere to close the Marker Manager dialog box 3 Select the wells containing the assays for a marker you configured in the previous procedure Note For easier selection of plate grid wells use the Ctrl and Shift keys to select wells individually or in groups See page 2 23 for more information 4 In the well inspector click the Use check box of the marker you want to add to the selected wells Note The detectors associated with the marker are automatically applied to the selected wells when the marker is placed in Use ee Marker Detector Reporter Task es E a CYP 2C19 2 Allele 2 WIC m Detectors for the i as PDAR CYP 2C9 2 f pom lele PDAR CYP 2C9 2 CE ii ai CYP 2cg e eee pacer on marker added to selected cass e s Wi 2 VIC wells of the plate CYP 2co allele 1 FAM i document _ s Use check box for PDAR CYP 2C9 2 selected 5 Ifnecessary repeat steps 3 through 4 to assign any remaining markers to the plate document 6 Configure the plate document with det
311. ment click stop e Programmed the plate document 2 Determine how far into the run the with the wrong thermal profile instrument has progressed 3 Based on the state of the run determine whether the plate can be re run 4 If necessary eject the plate and add the missing reaction component 5 If desired re run the plate by re creating the plate document 4 28 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide After the Run After the Run User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to save a plate document Ejecting a Plate View the instrument status icon to determine the plate document to use to eject the plate Opening and Closing the Instrument Status Action Instrument Tray 1 In the SDS software select Window gt the plate document connected to the instrument Connected to 2 In the plate document select the Instrument tab platename 3 Select the Plate Read or Real Time tab Click Open Close In the SDS software click E or select File gt New Click OK Select the Instrument tab gt z Disconnected Disconnected Select the Plate Read or Real Time tab Click Open Clase Saving the After the run you must either save the results to the SDS Enterprise Database or save Run Data the run data to the SDS 7900HT Document sds
312. most effective when NTCs are tasked Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 5 7 Chapter 5 Analyzing End Point Data Cluster Variations The SDS software graphs the results of an allelic discrimination run on a scatterplot contrasting reporter dye fluorescence Allele X R versus Allele Y R The software represents each well of the 384 well plate as a datapoint on the plot The clustering of these datapoints can vary along the horizontal axis Allele X vertical axis Allele Y or diagonal Allele X Allele Y This variation is due to differences in the extent of PCR amplification which could result from differences in initial DNA concentration The example below shows the variation in clustering due to differences in the extent of PCR amplification Setup Instrument Results cae fk eea al aby i Hs ff Pe Allele X Allele Y heterozygotes Allele Y homozygotes Legend Allele Allele vy Allele A27 E NTC w Undetermined Allele Y CYP 2C419 2 1 Outliers Allele X homozygotes No amplification 1 0 o 0 1 0 20 3 0 40 Allele X CYP 2C19 2 2 Figure 5 4 Common Datapoint Clusters Genotypic Segregation of Datapoint Clusters Figure 5 4 illustrates the concept of genotypic segregation of samples within the allele plot The plot contains four separate distinct clusters that represent the No Template Controls and
313. mple bars 6 31 X axis 6 31 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Index 3 Y axis 6 31 grey dividing line using 2 21 grid See plate grid gripper 7 36 guidelines absolute quantification assay development B 6 allelic discrimination assay development B 5 loading the Plate Handler 4 41 relative quantification assay development B 6 TaqMan probe design B 2 to B 3 H hand held bar code reader connections 1 10 location 1 7 specification 1 7 using 2 27 hard drive defragmenting 7 54 partitions 1 6 heated clamp 1 4 help background information 2 3 using the Sequence Detection Systems Software Online Help 2 3 hold adding to a method 3 19 hotkey combinations using 2 28 importing setup table data 2 16 A 2 imprecise pipetting 8 6 improper threshold setting 8 5 installing plate adapter 7 12 sample block module 7 6 SDS software 7 55 the operating system software 7 55 instrument 1 4 about 1 2 to 1 10 external components 1 3 firmware 1 14 internal components 1 4 loading plates 4 25 maintaining 7 3 optics system 1 5 status lights 2 5 supported runs and chemistries 1 2 troubleshooting 8 14 turning ON 2 4 instrument tray 4 44 opening and closing Automation Controller Software 4 44 opening and closing SDS software 4 29 replacing the plate adapter 7 12 IP Address A 22 irreproducibility causes 8 5 to 8 8 J Java Runtime Environment about 1 14 K keyboa
314. n all spectral data that consists of fluorescence from several sources including the background electronic signal the sample block the water inside the consumable and the plastic consumable itself Because the background signal can interfere with the precision of SDS data the 7900HT instrument has been engineered to minimize the background signal Additionally the SDS software also algorithmically eliminates the background signal from each fluorescent sample to maximize the instrument s sensitivity One of the following e Background plate included in a Spectral Calibration Kit and a centrifuge with plate adapter e Components for making a standard 384 or 96 well background plate ABI PRISM 394 or 96 Well Optical Reaction Plate An optical adhesive cover or ABI PRISM Optical Flat Caps Pipettor 100 uL with pipet tips Centrifuge with plate adapter e Components for making a TaqMan Low Density Array background plate Microfluidic card spectral calibration reagents Centrifuge Microfluidic card sealer 7 16 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Performing a Background Run Preparing the Background Plate or TaqMan Low Density Array Preparing a Background Card Preparing and Running a Background Plate Remove the background Low Density Array from its packaging and set it aside to warm to room temperature Remove the tube of background s
315. n any of the features described below see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide The Reason s for Change dialog box appears only if the SDS Enterprise Database is configured for auditing The auditing system monitors the creation modification and deletion of the SDS data contained by the database When the Reasons for Change dialog box appears do one of the following e Select a description for the change from the drop down list then click ox e Enter a description of the reason for the change in the field then click si a Reasons for change Reason For Change drop down list Atirinta AGPiato Cid Vetus New Value Default descriptions Calculation Error Enter the Reas oe Need to Change Threshold Custom description of the reason for the change Figure 6 1 Reasons for Change Dialog Box Options The Electronic Signature Verification dialog box appears only if the SDS Enterprise Database is configured to require electronic signatures The electronic signature system restricts and tracks user access of the SDS data contained by the database When the Electronic Signature Verification dialog box appears 1 In the User ID and Password fields enter your user name and password then click 2 After the software validates your user name and password it displays a message stating the success or failure of the signature
316. n of the SYBR Green 1 dye during a single cycle of a PCR 3 y r 7 4 1 5 ge I ar I When added to the reaction the SYBR Green 1 Dye binds non specifically to the hybridized dsDNA and fluoresces Dissociation O 0 O tn a 3 Denaturation complete the SYBR Green 1 Dye dissociates from the strand resulting in decreased fluorescence Polymerization Forward O O Primer A hm wll 5 3 PUTTIN a AUNRRERREAE AL 5 lif LLIN ALLL Mi LLL LLN VILL A Ah HII ENTREE Reverse Primer During the extension phase the SYBR Green 1 Dye begins binding to the PCR product Polymerization Complete UL LN ALLL iii LLLA LN ALLU AN ALLU AnH LLL LLIN LLU LLY BIEN IN wT AE TT LS 1 Aiii TENN Ai BENET N LLON Polymerization is complete and SYBR Green 1 Dye is completely bound resulting in a net increase in fluorescence Figure D 2 Binding Activity of the SYBR Green 1 Dye during the PCR Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide D 3 Appendix D Theory of Operation Real Time Data Analysis Kinetic Analysis Quantitative PCR The 7900
317. n reaction and plasmid DNA complementarity determining region III standards Cancer Res 58 1998 3957 3964 Hartel C Bein G Kirchner H and Kluter H A Human Whole Blood Assay for Analysis of T Cell Function by Quantification of Cytokine mRNA Scand J Immunol 49 6 649 654 1999 Higgins J A et al 5 nuclease PCR assay to detect Yersinia pestis In Process Citation J Clin Microbiol 36 1998 2284 2288 Hennig H et al Lack of evidence for Marek s disease virus genomic sequences in leukocyte DNA from multiple sclerosis patients in Germany In Process Citation Neurosci Lett 250 1998 138 140 Houng H H Hritz D and Kanesa thasan N Quantitative detection of dengue 2 virus using fluorogenic RT PCR based on 3 noncoding sequence J Virol Methods 2000 Apr 86 1 1 11 Kafert S J Krauter A Ganser and M Eder Differential quantitation of alternatively spliced messenger RNAs using isoform specific real time RT PCR Anal Biochem 269 210 213 1999 Kimura H Morita M Yabuta Y Kuzushima K Kato K Kojima S Matsuyama T and Morishima T Quantitative analysis of Epstein Barr virus load by using a real time PCR assay J Clin Microbiol 37 1 132 136 1999 Klein D Janda P Steinborn R Muller M Salmons B and Gunzburg W H Proviral load determination of different feline immunodeficiency virus isolates using real time polymerase chain reaction influence of mismatches on quantifi
318. nalysis The following options are available after the analysis Options Changing the Display Settings ccc cece eee eee eee 6 52 Pem COO oe hace iti eer ences auido cetyed dart e tn nas eos 6 52 Exporting Plate Document Data 2s cas ow heed naehewt awe toed bee dae eas 6 52 Saving the Results ne see below Saving the After an analysis the SDS software allows you to save the results as an SDS 7900HT Analyzed Study Multiple Plate Document sdm as a Multiple Plate Document T Save the analysis to an SDS 7900HT Multiple Plate Document sdm 1 Inthe SDS software select File gt Save As 2 Inthe Look in field of the Save As dialog box navigate to and select a directory for the software to receive the new file 3 Inthe File name field enter a file name for the file 4 Click _ se The software saves the plate document to the specified directory Saving the Study to the SDS Enterprise Database The SDS Enterprise Database does not store relative quantification studies SDS 7900HT Multiple Plate Documents Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 35 Chapter 6 Analyzing Real Time Data 6 36 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 6 3 Dissociation Curve Analysis Section 6 3 Dissociation Curve Analysis I This Section SOVSPVIOW uo o5 chy kikit iot 4055449 ERTER 55s AREER ee Rese ee 6 38 Berne T
319. ndent protein Quantitation kinase catalytic subunit of severe combined immune deficiency mice PNAS 94 2438 2443 1997 Bieche I et al Novel approach to quantitative polymerase chain reaction using real time detection application to the detection of gene amplification in breast cancer Int J Cancer 78 1998 661 666 Bieche I P Onody I Laurendeau M Olivi D Vidaud R Lidereau and M Vidaud Real time reverse transcription PCR assay for future management of ERBB2 based clinical applications Clin Chem 45 1148 1156 1999 Casteels K M et al Prevention of type I diabetes in nonobese diabetic mice by late intervention with nonhypercalcemic analogs of 1 25 dihydroxyvitamin D3 in combination with a short induction course of cyclosporin A Endocrinology 139 1998 95 102 Chiang P W Beer D G Wei W L Orringer M B and Kurnit D M Detection of erbB 2 amplifications in tumors and sera from esophageal carcinoma patients Clin Cancer Res 5 6 1381 1386 1999 Collins C et al Positional cloning of ZNF217 and NABC1 genes amplified at 20q13 2 and overexpressed in breast carcinoma Proc Natl Acad Sci U S A 95 1998 8703 8708 de Kok J B Ruers T J van Muijen G N van Bokhoven A Willems H L and Swinkels D W Real time quantification of human telomerase reverse transcriptase mRNA in tumors and healthy tissues Clin Chem 2000 Mar 46 3 3 13 8 Dittmer D C Stoddart R Renne V Linquist Step
320. ndler 8 17 8 20 turning ON the Applied Biosystems 7900HT Fast Real Time PCR System 2 4 U upgrading operating system software 7 55 SDS software 7 55 V viewing well information 2 23 views hiding 2 21 maximizing minimizing 2 22 resizing 2 21 2 22 showing 2 21 W warranty statement E 1 well inspector 2 31 Y Y Inter readout from the Standard Curve Plot 6 13 Z zooming allelic discrimination plot 5 14 plate grid wells 2 26 Zymark Twister Microplate Handler See Plate Handler Zymark Twister Software about 1 14 aligning the Plate Handler 7 41 to 7 48 closing 7 48 launching 7 41 testing the plate sensor switch 7 39 Index 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Science iScience To better understand the complex interaction of biological systems life scientists are developing revolutionary approaches to discovery that unite technology informatics and traditional laboratory research In partnership with our customers Applied Biosystems provides the innovative products services and knowledge resources that make this new Integrated Science possible Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web
321. ned to the well Each detector is represented by three text blocks that define the following information The name of the detector The task assignment of the detector for the well UNKN Unknown STND Standard NTC No Template Control The quantity assignment of the detector for the well For wells containing standards assign the quantity for the standard sample in initial copy number For all other wells assign the quantity value as 0 To assign more than one detector to a well repeat the detector definition text blocks for each detector There is no limit to the number of detectors that can appear in a well IMPORTANT All detectors that appear in this section must have been previously defined in the Detector Definitions section elements 4 6 A 10 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Setup Table Files Format for a single well Well number lt tab gt SDS Sample Name lt tab gt Detector name lt tab gt Detector task lt tab gt Detector quantity Detector task lt tab gt Detector quantity lt tab gt Detector name lt tab gt lt tab gt Detector name lt tab gt Detector task lt tab gt Detector quantity lt cr gt Example Example Code A 8 Allelic discrimination setup table files SDS Setup File Version 3 Output Plate Size 384 Output Plate ID 384N75822034 Number of Detectors 2 Detector Repo
322. nitors the creation modification and deletion of the SDS data contained by the database When the Reasons for Change dialog box appears do one of the following e Select a description for the change from the drop down list then click ox e Enter a description of the reason for the change in the field then click Reasons for change drop down list Resson For Changa Attribute AGPlato Cid vee Nena Wale Default descriptions Calculation Error Meed to Change Threshold Meed to Reanalyze Erie ihe Reaconion Change Example of a custom description for a change Figure 7 1 Reasons for Change Dialog Box Options Custom description of the reason for the change The Electronic Signature Verification dialog box appears only if the SDS Enterprise Database is configured to require electronic signatures The electronic signature system restricts and tracks user access of the SDS data contained by the database When the Electronic Signature Verification dialog box appears 1 In the User ID and Password fields enter your user name and password then click x 2 After the software validates your user name and password it displays a message stating the success or failure of the signature Click ok to continue You are attempting to change the for property x of Che curren Description of the action that requires a signature Enter your user name Enter your p
323. normal operating conditions the instrument laser is categorized as a Class 3B laser When safety interlocks are disabled during certain servicing procedures the laser can cause permanent eye damage and therefore is classified under those conditions as a Class 3B laser The Applied Biosystems 7900HT Fast Real Time PCR System complies with 21 CFR 1040 10 and 1040 11 as applicable The Applied Biosystems 7900HT Fast Real Time PCR System has been tested to and complies with the Radiation Control for Health and Safety Act of 1968 Performance Standard CFR 1040 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide xxi Safety and EMC Compliance Information Laser Safety Requirements Additional Laser Safety Information The Applied Biosystems 7900HT Fast Real Time PCR System has been tested to and complies with standard EN60825 1 Radiation Safety of Laser Products Equipment Classification Requirements and User s Guide To ensure safe laser operation e The system must be installed and maintained by an Applied Biosystems Technical Representative e All instrument panels must be in place on the instrument while the instrument is operating When all panels are installed there is no detectable radiation present If any panel is removed when the laser is operating during service with safety interlocks disabled you may be exposed to laser emissions in excess of the Class 3B rating
324. not stare directly into the beam DANGER de Class 3B rayonnement laser en cas d ouverture et d une neutralisation des dispositifs de securite Eviter toute exposition directe avec le faisceau DANGER Class 3B laser radiation present when open Do DANGER de Class 3B rayonnement laser en cas d ouverture Eviter toute exposition directe avec le faisceau DANGER Class 3B LED when open and interlock defeated Do not stare directly into the beam DANGER Class 3B LED when open Do not stare directly into the beam DANGER de Class 3B LED en cas d ouverture et d une neutralisation des dispositifs de securite Eviter toute exposition directe avec le faisceau DANGER de Class 3B LED en cas d ouverture Eviter toute exposition directe avec le faisceau CAUTION Moving parts ATTENTION Parties mobiles xvi Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide General Instrument Safety General Instrument Safety Nee PHYSICAL INJURY HAZARD Use this product only as specified in this document Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument Moving and N Oe PHYSICAL INJURY HAZARD The instrument is to be moved Lifting the and positioned only by the personnel or vendor specified in the applicable site Instrument preparation guide If you decide to lift or move the instrument after it has been instal
325. nspector Panel see page 2 12 Hides shows the Plate List Panel see page 2 12 H Hides shows the Plate Grid see page 2 12 H Hides shows the Table Pane see page 2 12 H Hides shows the System Raw Data Pane see page 2 12 Hides shows the Multicomponent Plot see page 2 12 A Hides shows the Amplification Plot see page 2 12 H Hides shows the Standard Curve Plot see page 2 12 Hides shows the Gene Expression Plot see page 2 12 Hides shows the Dissociation Plot see page 2 12 H Zooms the plate grid see page 2 12 Opens the Display Settings dialog box see page 2 12 2 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Using the SDS Software About the The message bar displays a variety of messages to indicate the status of the Message Bar instrument and software The following table summarizes all of the messages displayed in the Message bar Table 2 2 Status Messages Message Then the SDS software is Ready ready and awaiting instructions Collecting Data currently running a plate document Reanalyze data requesting analysis of plate document data The analysis settings for the plate document have been changed and the document requires reanalysis for them to take effect Analyzing data Progress bar completing the calculations for the current analysis The metered bar to the right of the message displays the progres
326. nt Volume uL TaqMan 2X Universal PCR Master Mix 500 Human cDNA 50 RNase DNase free water 450 Total Volume 1000 6 Tap the tube gently to mix 7 Pipette 100 uL of the reaction mix into each of the eight fill reservoirs 8 Continue with Preparing the Plate Document on page 7 33 7 32 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Preparing the 1 Plate Document 2 Verifying Instrument Performance Start the SDS software Click or select File gt New Configure the New Document dialog box Assay Absolute Quantification Container Select the appropriate plate format Template If running a e TaqMan RNase P 384 Well Instrument Verification Plate select 384 Well RNaseP Install Plate sdt e TaqMan RNase P Instrument Verification Plate standard 96 well plate select 96 Well RNaseP Install Plate sdt e TaqMan RNase P Fast 96 Well Instrument Verification Plate select 96 Well RNaseP Install Plate sdt e TGF B card select rnase p card template sdt Note If no plate document templates are available construct a Pure Dye plate document using the product insert from the Spectral Calibration Kit and the procedure on page 7 28 If desired enter the bar code information into the plate document a Click the Barcode text field b Remove the TaqMan RNase P Instrument Verification Plate or TGF B card from the packaging and scan its bar cod
327. nt has a heated clamp that provides optimal heat transfer and uniform heating during thermal cycling When the instrument tray loads a plate the clamp applies a downward pressure of 70 Ibs 31 8 kg onto the consumable During the run the clamp maintains a constant temperature of 105 C 3 C to prevent condensation within the consumable wells 1 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 79O0HT Instrument Interchangeable The 7900HT instrument features a Peltier based interchangeable sample block Thermal Cycler module based on the technology established in the GeneAmp PCR System 9700 Blocks thermal cycler The sample block module houses an internal Peltier heating cooling unit Figure 1 4 The sample block module is made of aluminum to provide an optimal thermal transfer rate between the block and the optical plate or the TaqMan Low Density Array ANTENY PHYSICAL INJURY HAZARD Hot Surface Use care when working around this area to avoid being burned by hot components Connection to the instrument Do Not Touch Circuitry Do Not Touch Sample block Heat sinks Do Not Touch Do Not Touch Block inside i instrument Top view Bottom view Figure 1 4 Components of the Interchangeable Thermal Cycler Block The interchangeable sample block module provides Wide temperature range 4 99 9 C Accuracy 0 25 C from 35 99 9 C Heat cool rate
328. o perform a dissociation Curve analysis do the following 1 Program the method for the absolute quantification experiment as explained on page 3 19 2 Add a temperature ramp to the method for dissociation curve analysis as explained on page 3 21 Database Configure the method of your plate document to collect data only in the necessary Considerations _ stages of the PCR Because of memory constraints the SDS Enterprise Database cannot save real time plate documents greater than 40 MB The file size for a real time run depends on the length of the run and the configuration and placement of the data collection icons Longer runs and runs configured to collect data at multiple stages of the method can be considerably larger than the 15 25 MB average Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 17 Chapter 3 Preparing a Run Programming Methods for Allelic Discrimination To maximize instrument throughput the SDS software does not provide the option to thermal cycle allelic discrimination plate documents Because allelic discrimination experiments are end point runs that do not require data collection during the PCR you can perform thermal cycling on a dedicated thermal cycler and then transfer to the 7900HT instrument for data collection and analysis If you want to thermal cycle the ee aise ek Then allelic discrimination plates on a designated thermal cycler go
329. ocument sds SDS 7900HT Template Document sdt absolute quantification AQ allele allelic discrimination AD amplicon Amplification plot analysis session annealing Application Program Interface API assay type auto increment value Glossary 2 A file used by the SDS software to store plate documentation locally on a computer hard drive A plate document is a virtual representation of a consumable plate used to contain samples and reagents during a sequence detection run During the run the software uses the plate document to record the operation of the instrument thermal cycling and data collection parameters organize and store the data gathered during the PCR process store the of the run data and record analysis and display options Plate document templates have the same format as the SDS 7900HT Documents sds but do not store raw data or bar codes The objective of an absolute quantification experiment is to accurately determine the absolute quantity of a single nucleic acid target sequence within an unknown sample The results of an absolute quantification experiment are reported in the same units of measure as the standard used to make them One of the variant forms of a gene at a particular locus or location on a chromosome The process by which two variants of a single nucleic acid sequence present in a population of individuals DNA samples is determined Single nucleotide polymorphism
330. of the following e Enter a file name for the plate document file e Enter or scan the bar code number for the plate into the field Note The SDS software does not require that the file name match the bar code of the corresponding plate 4 Click _ se The software saves the plate document to the specified directory Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 53 Chapter 6 Analyzing Real Time Data 6 54 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Maintaining the Instrument In This Chapter Motes Tor Database LIGN o odb hos ween se bind dca wkesn beckewebeensec ess 7 2 Recommended Maintenance Schedule 0 0 0 eee nee 7 3 Section 7 1 Maintaining the 7900HT Instrument sees 7 5 Replace toe Sample PGC 6 os pooh dn hee oan bog ears Gade oe 444s oe 25 7 6 Changing the Plate Adapter a beh 5 24 ebd0 548504045950 695 4 004 464 eRe ES 7 12 Decontaminating the Sample Block 0 0000 ccc cece eee 7 14 Performing a Background Rit o4644260004 4084045408 08 p48 ORE eS eS 7 16 Performing a Pure Dye Run gies 4054409 00454 e RSENS de EROS EDS EES EES 7 20 Adding Custom Dyes to the Pure Dye Set 0 0 0 0 0000 cee eee 7 27 Verifying Instrument Performance n n 56 bG soe bee wews boa chase wee os 7 30 Section 7 2 Maintaining the Plate Handler ccc wececccees 7 35 Automation Accessor
331. of the pipetting method Consult the manufacturer about the correct method of dispensing liquid volumes accurately from the pipettor For example some pipettors are designed to deliver the designated volume at the first plunger stop so blowing out the residue may cause error Also before using a new pipettor tip to serially dispense a master mix wet the tip once by drawing up some of the master mix and dispensing it back into the mix again Chemistries that have not been optimized may be susceptible to inconsistencies To maximize precision and reaction efficiency optimize the primer and probe concentrations of each individual assay used Refer to the ZagMan Universal PCR Master Mix Protocol PN 4304449 for specific information about optimizing probe and primer concentrations for TaqMan related chemistries For maximum precision the PCR master mix must be mixed to uniformity After all reaction components are added to master mix it should be vortexed for 4 5 seconds before aliquoting it to the wells of the plate Any dilutions performed during the assay should also be vortexed Air bubbles in the wells can refract and distort the fluorescent signals Ideally the reagents would be applied to the wells using a pipetting technique that does not form air bubbles However if a plate does contain air bubbles they can usually be removed by swinging tapping or briefly centrifuging the reaction plate If PCR reagents splash the undersi
332. oftware Related Files and Formats 0 00 0 cc eee eens 1 15 Managing Sequence Detection System Data 0 0 0 0 0 0 cee eee 1 17 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 13 Chapter 1 Product Overview Software Components Components of The Applied Biosystems 7900HT Fast Real Time PCR System uses a suite of the SDS Software applications to set up run and analyze experiments completed on the 7900HT Suite instrument Table 1 3 Standard SDS Software Software Function SDS Software e Constructs and edits plate document files e Performs initial and end analysis of raw data from allelic discrimination relative quantification and absolute quantification runs e Saves prints and exports run data Java Runtime Environment Additional files and software used to run the SDS software IMPORTANT Do not delete the Java Runtime Environment files These files are crucial to the operation of the SDS software 7900HT Instrument Firmware Controls the most basic operations of the 7900HT instrument e Controlled by commands sent from the computer e Links the commands issued by the SDS software and the operation of the 7900HT instrument Table 1 4 Software for Automated or SDS Enterprise Database Operation Software Function Automation Controller e Controls and coordinates the action of the 7900HT Software instrument and the automation mod
333. ogical series of measurements of the fluorescence resulting from the reactions Examples of real time chemistry include absolute quantification relative quantification and dissociation curve analysis 1 2 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 1 1 Getting to Know the Hardware Section 1 1 Getting to Know the Hardware In This Section 7900HT Instrument 0 0 0000000 ec cece eee eens 1 4 Ce a eka oe hone oe ed ohn ee Oe oe EEEE TETIERE AET aes 1 6 Har Cado ete porosi hed ook harr deb see eees ee kee ee oka 1 7 Zymark Twister Microplate Handler 0 0 0 0 cee 1 8 Compatible Consumables rsrirrerrerritkidi i oe HELO ERRI Gow eGo Hoes ess 1 9 Instrument Connections a5 a s 456 5k es bo Oe ee ESKER ESSE ED EERO OER OHS 1 10 Instrument The Applied Biosystems 7900HT Fast Real Time PCR System consists of the Components components shown in Figure 1 2 Microsoft Windows 7900HT Instrument Compatible Computer see page 1 4 see page 1 6 Science GR2009 Fixed Position Zymark Twister Extended Hand Held Bar Bar Code Reader Microplate Handler Capacity Stacks Code Reader see page 1 7 see page 1 8 see page 1 7 see page 1 7 Figure 1 2 Components of the Applied Biosystems 7900HT Fast Real Time PCR System Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 3 Chapte
334. old data are the basis for generating gene expression values For any PCR experimental error may cause some wells to amplify insufficiently or not at all These wells typically produce C values that differ significantly from the average for the associated replicate wells If included in the relative quantification calculations these outliers can potentially result in erroneous measurements To ensure precise relative quantification replicate groups must be carefully scrutinized for outlying wells before the analysis The software provides options for removing outliers automatically see page 6 19 or manually see page 6 49 The final stage in the analysis is the computation of gene expression values from the C data The mathematical process for deriving relative quantification values is briefly described on page D 8 For detailed information on the derivation of the AAC equation relative quantification data transformations or other aspects of 5 nuclease chemistry in relation to relative quantification Applied Biosystems recommends the following references e For information on performing relative quantification using 5 nuclease chemistry on 7900HT instruments see ABI PRISM 7700 Sequence Detection System User Bulletin 2 Relative Quantification Of Gene Expression PN 4303859 RO Manager Software User Guide PN 4351670 The bibliography of this document for a list of technical papers and research e For information on the d
335. olson D Balducci Silano P L Levine M A Francis G L and Tuttle R M Quantitative reverse transcription PCR measurement of thyroglobulin mRNA in peripheral blood of healthy subjects Clin Chem 45 6 Pt 1 785 789 1999 Yajima T et al Quantitative reverse transcription PCR assay of the RNA component of human telomerase using the TaqMan fluorogenic detection system Clinical Chemistry 44 1998 2441 2445 Yamada Y N Itano H Narimatsu T Kudo S Hirohashi A Ochiai A Niimi M Ueda and K Kimata 1999 Receptor for hyaluronan mediated motility and CD44 expressions in colon cancer assessed by quantitative analysis using real time reverse transcriptase polymerase chain reaction Jpn J Cancer Res 90 987 992 Yoneyama H A Harada T Imai M Baba O Yoshie Y Zhang H Higashi M Murai H Asakura and K Matsushima 1998 Pivotal role of TARC a CC chemokine in bacteria induced fulminant hepatic failure in mice J Clin Invest 102 1933 1941 Zhang A W Hartman G L Curio Penny B Pedersen W L and Becker K B Molecular Detection of Diaporthe phaseolorum and Phomopsis longicolla from Soybean Seeds Phytopathology 89 9 796 804 1999 Allelic Breen G Harold D Ralston S Shaw D and St Clair D Determining SNP allele Discrimination frequencies in DNA pools Biotechniques 2000 Mar 28 3 464 S 470 Fujii K Y Matsubara J Akanuma K Takahashi S Kure Y Suzuki M Imaizumi K linuma O Sa
336. olution from the freezer to thaw When the solution has thawed vortex the tube Load the background solution into the background Low Density Array loading 100 uL of solution per fill reservoir Centrifuge the Low Density Array according to the procedures in Centrifuging the TaqMan Low Density Arrays on page 4 16 Seal the Low Density Array according to the procedures in Sealing the TaqMan Low Density Arrays on page 4 19 Continue with Creating a Plate Document for the Background Run below Choose from the following If Then using a background remove the plate from the freezer and allow it to thaw to plate from a Spectral room temperature Calibration Kit creating a 1 Pipette deionized water to each well of the plate background plate f using a standard 384 well plate add 20 uL per well lf using a standard 96 well plate add 50 uL per well 2 Seal the plate using an optical adhesive cover or optical flat caps 2 Briefly centrifuge the background plate 3 Continue with Creating a Plate Document for the Background Run below Creating a Plate Document for the Background Run Preparing a Background Plate Document Start the SDS software 2 Click or select File gt New 3 Configure the New Document dialog box e Assay Select Background e Container Select the appropriate plate format e Template Select Blank Template
337. omputer Problems Observation Possible Cause Recommended Action Computer is slow when analyzing data opening or closing dialog boxes and other software processes Hard drive is fragmented Defragment the hard drive as explained on Defragmenting the Hard Drive on page 7 54 Hard drive is almost full Remove or archive existing data files as explained on Archiving SDS Files on page 54 The computer will not logon to the Windows Operating System Logon window does not appear Restart the computer and logon to your computer You are not logged on as the Administrator 1 Logoff of your computer 2 Logon again as the Administrator After the above solutions have been tried the problem is still not fixed Contact Dell for troubleshooting the computer hardware or OS The computer will not boot up at all Cables are not connected or are not seated properly Check the cables The boot disk is corrupted 1 Boot directly off of the Windows NT Operating System Installation CD 2 Boot off of the emergency disk 3 Reload the Windows NT Operating System from the CD After the above solution has been tried the problem is still not fixed Contact Dell for troubleshooting the computer hardware Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Zymark Twister Microplate Handler and Fixed P
338. on Curve tab as explained on page 6 44 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 41 Chapter 6 Analyzing Real Time Data Determining T Values for the Analyzed Run Viewing Analyzed The SDS software displays the results of the dissociation curve analysis within the Dissociation Dissociation Curve tab of the plate document The tab displays the analyzed data in a Curve Data graph of the negative of the derivative R versus temperature T that visualizes the change in fluorescence at each temperature interval during the ramp Note The plot displays data from the selected wells of the plate grid If you do not see dissociation curve data select the wells of the plate grid containing the SYBR Green dye reactions Figure 6 15 illustrates the components of the Dissociation Plot Setup Instrument Results Dissociation Curve 4 Dissociation Curve tab Dissociation Plot fed 5 000 E 1 Dissociation Plot 4 000 E 1 3 000 E1 Derivative 2 000 E1 Dissociation curves 1 000 E 1 0 000 Tm display and slider Temperature Detector SYBR v Plot Derivative 7 Step Stage 6 step 1 Step drop down list Plot drop down list Detector drop down Figure 6 15 Elements of the Dissociation Plot e Dissociation plot The plot displays data from the selected wells in the plate grid Note The properties of the Dissociation Plot are adjustabl
339. on Controller Software Running Plates Using the Automation Controller Software User Access There is no access requirement All users can run plates that have been saved to the Requirement SDS Enterprise Database Loading Plates onto the Plate Handler Guidelines Observe the following guidelines when loading plates onto the Plate Handler e Before loading plates onto the Plate Handler make sure that for each plate the associated plate document has been added to the plate queue or is contained in the SDS Enterprise Database the plate has been sealed using an optical adhesive cover e Load the plates into the Plate Handler stacks in any order The software reads the bar code of each plate before it is run and matches the plate document and method with the plate The Automation Controller Software can run batches of up to 84 plates in a single session 21 plates stack e Orient the plates inside the stacks so that well Al e of each plate corresponds to the locations shown in Figure 4 11 e Do not place plates in the output stack X The Plate Handler arm uses the empty stack to store plates after it runs them e If loading Low Density Array s onto the Plate Handler Load no more than 20 cards per stack Pack stacks of cards tightly The Plate Handler may fail to detect a misaligned card correctly when loading it Zymark Twister Microplate Handler top view Bar code Well A1 Figure 4 11 Plat
340. on over the sealer s insert plate The Low Density Array s fill reservoir end should be the end closest to the arrows etched in the base of the sealer b Insert the Low Density Array foil side up on top of the insert plate 3 Gently push the Low Density Array until it is seated securely in the insert plate Note Two metal pins on the sealer s insert plate project into corresponding holes molded into the Low Density Array s carrier to provide mechanical alignment for the sealing process When properly seated the Low Density Array s foil surface should be level with the base of the sealer Note Four spring clips ensure that the Low Density Array is held in the proper position Sealing a Card 1 Push the carriage across the base of the sealer in the direction of the arrows Use one firm smooth motion until you reach the end of the sealer N oF el The sealer has mechanical stops at both ends to prevent the carriage from coming off Do not use excessive force or speed when pushing the carriage Using force will not increase the sealing quality but only cause wear to the sealer End position Move in this direction 4 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Preparing TaqMan Low Density Arrays for Use 2 Remove the sealed Low Density Array by grasping its sides and lifting it off the sealer s insert plate Note In the middle of the sealer s inser
341. on page 7 24 Analyzing the Pure Dye Run on page 7 25 7 26 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Adding Custom Dyes to the Pure Dye Set Adding Custom Dyes to the Pure Dye Set When to Perform The Applied Biosystems 7900HT Fast Real Time PCR System can be used to run assays designed with custom dyes dyes not manufactured by Applied Biosystems However before using custom dyes with the 7900HT instrument you must create and run a custom pure dye plate Materials e ABI PRISM 384 or 96 Well Optical Reaction Plate or Optical 96 Well Fast Required Thermal Cycling Plate e An optical adhesive cover or ABI PRISM Optical Flat Caps e Custom Dye s e Pipettor 100 uL with pipet tips e Centrifuge with plate adaptor Creating a_ Note The purpose of the custom pure dye plate is identical to that of an ABI PRISM Custom Pure Dye Pure Dye Plate The SDS software uses the custom plate to create a spectral standard Plate for multicomponenting the custom dye 1 Prepare a microplate with a dilution series of the custom dye 2 Start the SDS software 3 Create an allelic discrimination plate document and run the dilution series plate Note It is not necessary to configure detector sample and method information for the dilution series plate document The purpose of the run is to establish the correct working concentration for the dye by viewing the intensity of the raw spectra p
342. on to Step 8 Applying Sample and Plate Information on page 3 25 the 7900HT instrument follow the procedure below Performing Thermal Cycling of Allelic Discrimination Plates on the 7900HT Instrument To perform the thermal cycling and the plate read using the 7900HT instrument run the plate first as a real time plate document and then again as an allelic discrimination plate document as explained below IMPORTANT Follow the procedure below only if you intend to perform the PCR on the 7900HT instrument Otherwise perform the PCR on a dedicated thermal cycler and then transfer the plate to the 7900HT instrument for data collection 1 Start the SDS software 2 Create a real time plate document for absolute quantification as described on page 3 8 Note It is not necessary to configure the plate document with detectors 3 Program the plate document method with the thermal cycling times and temperatures for your protocol as described in Programming Methods for Absolute or Relative Quantification on page 3 19 4 Save the plate document as explained on page 4 24 IMPORTANT Do not save the real time plate document to the database The database cannot store plate documents of different run types with the same bar code 5 Run the plate using the real time plate document as described on page 4 25 Note Although large the real time file may be helpful in diagnosing and troubleshooting the experiment lat
343. one acknowledging that it has read the code Am LASER HAZARD Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots Never look into the laser beam Remove jewelry and anything else that can reflect the beam into your eyes Protect others from exposure to the beam mit PECYOO1DL3 20 30 cm After the gun has read the bar code the software automatically populates the selected field with the alphanumeric equivalent of the bar code 4 Click to close the Document Information dialog box Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 27 Chapter 2 Getting Started Lesson 5 Using Contextual Menus Overview The SDS software features contextual menus as a timesaving device to provide access to the commands for an associated view or pane To access a contextual menu move the pointer over a pane or view of interest then right click The menu appears at the location of the pointer Le SOS 2k Fie Edt Vew Toots heiren Anayo Ma ee a Copy Wells Paste Wells Contextual menu Clear Wells Fill Dewy Fill Fight p A fa w fa ig Li Hide Pane Save Grid to Image File Display Settings Figure 2 7 Contextual Menu of the SDS Software All contextual menus provide the following common commands Table 2 4 Standard Commands of the Contextual Menu Command Resu
344. onsumables and Disposables for the 7900HT Instrument Part No Description Quantity 96 Well Optical Reaction Plates 4306737 ABI PRISM 96 Well Optical Reaction Plate with Barcode 20 Plates code 128 4326659 ABI PRISM 96 Well Optical Reaction Plate with Barcode 500 Plates code 128 25 Pack Includes 25 of PN 4806737 ABI PRISM 96 Well Optical Reaction Plates with Barcode 4314320 ABI PRISM 96 Well Optical Reaction Plate with Barcode 100 Plates code 128 and ABI PRISM Optical Adhesive Covers 100 Covers Includes 100 ABI PRISM Optical Adhesive Covers PN 4811971 and 5 of PN 4806737 ABI PRiISM 96 Well Optical Reaction Plates with Barcode 4312063 MicroAmp Splash Free Support Base for 96 Well Reaction 10 Bases Plates Optical 96 Well Fast Plates 4346906 Optical 96 Well Fast Thermal Cycling Plate with Barcode 20 Plates code 128 4312063 MicroAmp Splash Free Support Base for 96 Well Reaction 10 Bases Plates Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Instrument Maintenance and Verification Instrument Maintenance and Verification The following sequence detection kits and reagents are used to perform routine maintenance on and verify the function of the Applied Biosystems 7900HT Fast Real Time PCR System For more information about the use of the kits below see Chapter 7 Maintaining the Instrument
345. optical side of the Low Density Array After removing the Low Density Array from its packaging water condenses on the reaction wells optical side of the Low Density Array The Low Density Array may not have come to room temperature before being removed from its packaging by lightly tapping or blowing on the reaction wells Room temperature pressurized nitrogen or an air blower may be used IMPORTANT Be sure to remove all dust and particulates The reaction wells optical side of the Low Density Array must be free of dust and particulates especially fluorescent particulates Remove condensation by lightly blowing on the reaction wells Room temperature pressurized nitrogen or an air blower may be used IMPORTANT Be sure to remove all water condensation The exterior surface of the reaction wells optical side of the Low Density Array must be free of water condensation Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide TaqMan Low Density Array Table 8 6 Troubleshooting the TaqMan Low Density Array Observation Possible Cause Recommended Action After pipetting too little PCR reaction mixture has gone into the fill reservoir After pipetting some of the PCR reaction mixture leaks out of the vent port in the fill reservoir The PCR reaction mixture was not correctly pipetted into the fill reservoir Care must be taken
346. or sample block could be contaminated e Characteristic signal shape Do the samples peak at the expected wavelengths For example samples containing only FAM dye labeled TaqMan probes should not produce raw fluorescence in the peak wavelength of the VIC dye component A signal present in wells that do not contain the dye could indicate that the sample master mix or well contains contaminants e Signal Plateaus Do any of the signals plateau Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 8 13 Chapter 8 Troubleshooting Software and 7900HT Instrument Troubleshooting Software and Computer Problems Table 8 4 Troubleshooting Software and Computer Problems Observation Possible Cause Recommended Action Unable to finish installation Install program appears to be frozen SDS Software version 2 2 does not function after the installation of SDS Software version 2 2 1 Installer dialog box is hidden from view SDS Software version 2 2 is automatically removed during the installation 1 Press and hold Alt 2 Press Tab until the Installer icon is selected 3 Release both keys The installer dialog box returns to the front of the desktop Reinstall the SDS Software version 2 2 SDS software will not start The software crashes freezes th
347. or example Select File gt Open gt Spot Set Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical Safety alert words also appear in user documentation For more information see Safety Alert Words on page xiv Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide xi Preface How to Obtain More Information Related See the following related documents for more information on the topics in this guide Documentation e Applied Biosystems 7900HT Fast Real Time PCR System Quick Starts Provide brief procedures for performing application specific tasks on the 7900HT instrument Sequence Detections Systems Software Online Help Describes the Sequence Detection Systems SDS Software and provides procedures for common tasks SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide Provides information for database administrators who will be maintaining the SDS Enterprise Database and provides information for systems integrators who will be working with the SDS Enterprise Database API Note
348. or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems This service is free and available 24 hours a day To obtain MSDSs 1 Go to www appliedbiosystems com click Support then click MSDS Search 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following e Open To view the document e Print Target To print the document e Save Target As To download a PDF version of the documen
349. or the Alignment Perform the following procedure if the Applied Biosystems 7900HT Fast Real Time PCR System is moved or the Zymark Twister Microplate Handler becomes misaligned Symptoms that the Plate Handler is out of alignment include Excessive downward movement of the Plate Handler arm the arm grinds when grasping or releasing plates The Plate Handler arm collides with the plate stacks The Plate Handler arm releases plates above the bottom of the plate stacks Reaction plates tip or tilt when placed into the instrument tray by the arm Remove the covers for the fixed position bar code reader and the underlying platform Fixed position bar code reader and underlying platform covers Loosen the three black thumbscrews on the platform connecting the 7900HT instrument and the Plate Handler base m Black thumbscrews ba AR Move the instrument tray to the out position a Start the Automation Controller Software If an error dialog box appears reading Machine calibration values are not valid Please refer to documentation for calibration process click OK b Click Open Close The 7900HT instrument moves the instrument tray to the out position perpendicular to the instrument c Select File gt Exit The software quits the Automation Controller Software 4 Select str gt Programs gt amp Zymark Twister
350. ormation on the fluorogenic 5 nuclease assay real time data collection or the mathematical transformations of sequence detection data 6 16 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Overview Algorithmic Manipulation of Raw Data Multicomponenting Setting the Threshold and Calling Threshold Cycles Outlier Removal Generating Gene Expression Values The SDS software can analyze raw data immediately after a relative quantification run is complete The term raw data refers to the spectral data between 500 nm to 660 nm collected by the software during the real time run During the analysis the software automatically applies several mathematical transformations to the raw data to generate a more direct measure of the relationship between the spectral changes in the unknown samples The first mathematical transformation involves the conversion of the raw data expressed in terms of Fluorescent Signal vs Wavelength to pure dye components using the extracted pure dye standards At the same time the software also determines the contribution of each dye in the raw data using the multicomponent algorithm After multicomponenting the baseline and threshold values must be set for each detector in the study The software allows you to set the baseline and threshold values manually see page 6 49 or automatically see page 6 19 The Cy values computed using the baseline and thresh
351. osen the sample block locking bolt Sample block locking bar Sample block locking bolt 7 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Replacing the Sample Block 9 Loosen the thumbscrew securing the sample block locking bar to the instrument chassis may be a 5 32 inch hex bolt on some instruments thumbscrew 11 Remove the sample block from the instrument a Rotate the release lever at the base of the sample block 90 degrees b Being careful not to damage the heat sinks on the bottom of the sample block slide the sample block out of the instrument and place it on a clean level surface Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 9 Chapter 7 Maintaining the Instrument Replacing the IMPORTANT Before changing the sample block perform all required upgrades to Sample Block the SDS software and instrument firmware Failure to upgrade the software can render the instrument inoperable or result in damage to instrument components 1 Load the sample block into the instrument compartment a Being careful not to damage the heat sinks on the bottom of the sample block rest the sample block on the metal runners on either side of the instrument bay b Carefully slide the sample block into the instrument until the front of the block is flush with the rear of the locking bar c After it is seated firmly
352. osition Bar Code Reader Zymark Twister Microplate Handler and Fixed Position Bar Code Reader Automation Accessory Troubleshooting Table Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Table 8 5 Troubleshooting the Automation Accessory Observation Plate Handler emits grinding noise when picking up or putting down plates Plate Handler arm contacts racks when retrieving or stacking plates The Plate Handler arm releases plates awkwardly into the plate racks Reaction plates tip or tilt when placed into the instrument tray by the Plate Handler arm Plate Handler fails to sense or grasp plates Plates stick to the gripper fingers of the Plate Handler arm Plate Handler does not restack plates in original locations Fixed position bar code reader not reading plate bar codes Possible Cause Recommended Action Vertical offset too low Plate detector switch set too high Plate Handler rotary offset is incorrect or vertical offset is too low Re align the Plate Handler as explained in Aligning the Plate Handler on page 7 41 Plate sensor switch not adjusted properly Adjust the plate sensor switch as explained in Adjusting the Sensitivity of the Plate Sensor Switch on page 7 37 Gripper pads on the fingers of the Plate Handler arm are worn or dirty Change the gripper pads as explained in Cleaning and Replacing Gripp
353. ote The contents of the exported data vary depending on the type of plate document e Setup Table The exported file contains the contents of the table pane of a plate document before analysis The contents of the exported data varies depending on the type of plate document used to produce it See page A 4 for a detailed description of the Setup Table file 5 To export data from e All wells of the plate document Select the All Wells radio button e Selected wells of the plate grid Select the Selected Wells radio button 6 In the files of type drop down list select the appropriate format for the exported data 7 Click the File name text box and enter a name for the exported file 8 Click Beat The software saves the exported data to the designated location Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 17 Appendix A Software Reference Operating the SDS Software from a Command Line Overview The SDS software allows you to use batch files to analyze and export results from the command line Use of the command line interface is intended for advanced users such as systems administrators bioinformaticians and laboratory network administrators who choose to drive the application using a scripting language Note If you are unfamiliar with Microsoft DOS Applied Biosystems recommends running the application from the user interface Running the 1 Inthe desktop double clic
354. otide Synthesis C 6 To order custom oligonucleotides e Visit the Applied Biosystems Online Store http store appliedbiosystems com or e Email Applied Biosystems with your order OligosUS appliedbiosystems com Table C 4 Custom Oligonucleotides Part Description Number p TaqMan MGB Probes 5 Fluorescent label 6 FAM VIC or TET 4316034 5 000 6 000 pmols 4316033 15 000 25 000 pmols 4316032 50 000 100 000 pmols TaqMan Probes 5 Fluorescent label 6 FAM VIC or TET 3 label TAMRA 450025 5 000 6 000 pmols 450024 15 000 25 000 pmols 450023 50 000 100 000 pmols Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Custom Oligonucleotide Synthesis Table C 4 Custom Oligonucleotides Part Number Description Sequence Detection Primers 4304970 Minimum 4 000 pmols purified for sequence detection 4304971 Minimum 40 000 pmols purified for sequence detection 4304972 Minimum 130 000 pmols purified for sequence detection Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Appendix C Kits Reagents and Consumables C 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Theory of Operation In This Appendix Fluorescent Based Chemistries 0 0 0 00 ccc cece ees Real Time Data Analysis
355. ous control sequence Endogenous control targets are typically constitutive RNA or DNA sequences that are present at a statistically consistent level in all experimental samples By using the endogenous control as an active reference the data from the amplification of a messenger RNA mRNA target can be normalized for differences in the amount of total RNA added to each reaction Examples of common endogenous controls are GAPDH 18S rRNA and B Actin The endogenous control can be amplified independently of the target sequences in separate wells on the reaction plate as singleplex or non multiplex reactions or in the same well as multiplex reactions IMPORTANT For non multiplex experiments the reactions amplifying the endogenous control must be located on the same plate as the target assays IMPORTANT To generate a standard deviation for the relative quantity value of a target each plate must contain usable data from at least two replicates of the target and endogenous control All relative quantification experiments require data from a calibrator sample During analysis the SDS software calculates gene expression levels in samples relative to the level of expression in a calibrator sample Thus the calibrator plays an integral part in the calculation because it is used as the basis for the comparative results Examples of possible calibrator samples include e A zero time point sample in a time course experiment e An untreated samp
356. owing analysis of the study data Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Overview Automatic Outlier Removal About Outlying For any PCR experimental error may cause some wells to amplify insufficiently or Replicate Wells notat all These wells typically produce Cy values that differ significantly from the average for the associated replicate wells If included in the calculations the C values from these wells indicate an incorrect level of relative gene expression by skewing the average for the replicate group About the The SDS software offers algorithmic identification and removal of outlying data for Automatic Outlier studies consisting of replicate populations of three or more wells The statistical Removal method used by the software is based on Grubbs outlier removal also known as the Maximum Normalized Residual Test which permits the exclusion of a single outlier in a population consisting of as few as three replicates The software applies the Grubbs tests at different stages in the AAC calculation depending on the type of endogenous control used in the study For non multiplex studies the software removes outliers from replicate groups immediately after calculating threshold cycles C For multiplex studies the software applies the algorithm following the AC calculation Also if an apparent outlier is within 0 25 C s of the mean for the associated replicate group the so
357. pairs and replacements under this Warranty will be performed by Applied Biosystems on site at the Customer s location at Applied Biosystems s sole expense No agent employee or representative of Applied Biosystems has any authority to bind Applied Biosystems to any affirmation representation or warranty concerning the Instrument that is not contained in Applied Biosystems s printed product literature or this Warranty Statement Any such affirmation representation or warranty made by any agent employee or representative of Applied Biosystems will not be binding on Applied Biosystems Applied Biosystems shall not be liable for any incidental special or consequential loss damage or expense directly or indirectly arising from the purchase or use of the Instrument Applied Biosystems makes no warranty whatsoever with regard to products or parts furnished by third parties Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide F 1 Appendix E Limited Warranty Statement This Warranty is limited to the original location of installation and is not transferable THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS E 2 Applied Biosystems 7900HT Fast Real
358. pecifications Applied Biosystems will repair or replace the Instrument so that it meets the Specifications at Applied Biosystems expense However if the thermal cycling module becomes damaged or contaminated or if the chemical performance of the Instrument otherwise deteriorates due to solvents and or reagents other than those supplied or expressly recommended by Applied Biosystems Applied Biosystems will return the Instrument to Specification at the customer s request and at the customer s expense After this service is performed coverage of the parts repaired or replaced will be restored thereafter for the remainder of the original Warranty Period This Warranty does not extend to any Instrument or part which has been a the subject of an accident misuse or neglect b modified or repaired by a party other than Applied Biosystems or c used in a manner not in accordance with the instructions contained in the Instrument User s Manual This Warranty does not cover the customer installable accessories or customer installable consumable parts for the Instrument that are listed in the Instrument User s Manual Those items are covered by their own warranties Applied Biosystems obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct those failures of the Instrument to meet the Specifications of which Applied Biosystems is notified prior to expiration of the Warranty Period All re
359. placing the Sample Block on page 7 10 Runa background plate to confirm that the contamination has been removed Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 15 Chapter 7 Maintaining the Instrument Performing a Background Run When to Perform Purpose of Background Runs About the Background Component Materials Required Applied Biosystems recommends running a background plate monthly or as often as necessary depending on instrument use A background run measures the ambient fluorescence in a background plate or Low Density Array containing deionized water During the run the 7900HT instrument performs a continuous scan of the plate for 2 minutes at 60 C Afterwards the SDS software averages the spectrum recorded during the run and extracts the resulting spectral component to a calibration file The software uses the calibration file during subsequent runs to remove the background signal from the run data Because the background signal can change with instrument age Applied Biosystems recommends regenerating the Background component calibration every month Note Background runs can also be used to detect and troubleshoot sample block contamination Fluorescence collected by the Applied Biosystems 7900HT Fast Real Time PCR System includes a background component a fluorescent signal that is inherent to the system The background component is a composite signal found i
360. plate adapter see page 7 12 A Row N Run a background plate to check the sample block for contamination see page 7 16 If you are changing block formats or installing a new block also do the following IMPORTANT Perform pure dye and background runs after replacing the sample block even if you are replacing a sample block module with another block of the same format 6 Runa pure dye plate or microfluidic pure dye card to create the pure spectra calibration values for the new format see page 7 20 7 Run the appropriate TaqMan RNase P Instrument Verification Plate or a TaqMan Low Density 7900HT Installation Array TGF B card to confirm the proper operation of the sample block see page 7 30 If you are using an Automation Accessory also do the following 8 If changing consumable formats for example when replacing a Standard 384 Well Block with a Standard 96 Well Block a Fast 96 Well Block or a 7900HT System TaqMan Low Density Array Upgrade adjust the plate sensor switch on the Plate Handler arm for the new plates see page 7 37 9 Align the Zymark Twister Microplate Handler fixed position bar code reader for the new plate format see page 7 41 IMPORTANT You must align the Plate Handler to only the Instrument position Zymark position 2 as explained on pages 7 41 to 7 44 10 Align the fixed position bar code reader for the new plate format see page 7 49 7 6 Applied Biosystems 7900HT Fast Real
361. plate in terms of detectors The well marker information consists of two sections the Well Detector List Header and the Well Detector Definition List 10 Well Detector List Header Description This line contains the column headings for the well marker information section of the setup table file that make the file easier to edit using a program such as Microsoft Excel Format Well lt tab gt Sample Name lt tab gt Detector lt tab gt Task lt tab gt Quantity lt tab gt Detector lt tab gt Task lt tab gt Quantity lt cr gt Example Example Code A 7 Well Sample Name Detector Task Quantity Detector Task Quantity 11 Well Detector Definition List Description This section defines the contents of the plate wells The setup table file must contain a definition for each well used on the plate Each well definition list consists of one string of characters terminated by a lt cr gt The definition consists of three main functional divisions e Well number The first tab delimited text block defines the number of the well on the plate Well numbers start at 1 for well A 1 upper left corner of the plate and increases from left to right and from top to bottom The wells must be listed in order 1 2 3 e Sample name The second text block defines the name of the sample assigned to the well e Detector assignments The remaining tab delimited text blocks for the well definition define the detectors assig
362. plied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 5 Chapter 2 Getting Started 2 6 Table 2 1 Instrument Status Lights Light Appearance Status Action Solid The 7900HT instrument has Power off the instrument wait rs detected a fatal problem for 30 sec and then restart id Note If the problem persists contact Applied Biosystems Red Technical Support Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Using the SDS Software Using the SDS Software Starting the 1 Do one of the following Software e Select gstat gt amp Programs gt amp Applied Biosystems gt amp SDS 2 2 1 gt ESDS 2 2 1 e Double click Dp SDS 2 2 1 on the computer desktop If you are using an SDS Enterprise Database on your local area network the software will automatically prompt you to log in 2 Database Only In the Login dialog box enter your user name and password and click ox Note When working with a database the SDS software requires that all users have a user account and password see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide PN 4351669 for more information on assigning and modifying user accounts The computer starts the SDS software and attempts to establish communication with the 7900HT instrument If the connection is successful the software d
363. ponding plate 4 Click Save The software saves the plate document to the specified directory 5 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Analyzing Real Time Data In This Chapter Notes for Database Users 0 00 ccc eects 6 2 Real Time Runs on the 7900HT Instrument 000002 eee 6 3 Section 6 1 Absolute Quantification cc ccc cece cece cree c neces 6 5 oes See ee ee een ee ee ee ee eee ee eee eee eee ee re ee ee 6 6 Were WA riers 54 4b 5 5g 4 e EFA ee 6 8 Pe I kk eg ds 4k es oS 4 SoS EEE SO EAA 6 9 penis ie Ron Das oe ge ao ed ee oe pee oe he ee eK eee Re OS 6 10 Analyzing the Ac oo ko 49 bee 54 Hb SP ON OE eRe 4S eb Oeste O ROR ee Os 6 11 le OR gn 24 Fe 86 Oh ee Ha 4 hese od E 45 be des 6 13 Pa ke ee os a ee a 8 a 2 oon ee 6 14 Section 6 2 Relative Quantification cc cece cece ec cere cece eees 6 15 Donn o so os 5 PP Ge ea 84 OSS RE Shes Oo eee eee 6 16 Essential Experimental Components 0 00 000 eee eens 6 20 hie oh on E E eo A E eh eo eek oe ee eee 6 21 a LO d EE ae de ee oe ee oe eho 4044 45405 6 22 Options for Analyzing Relative Quantification Data 04 6 23 Re E E oo oe og 4 bee Come nee Jaros ee eee ees 6 23 Analyzing TY sssri ny ok pa eee 8s OE SEND OA OE ROR OER oes A 6 26 yiewine ROWS e rastereti rr d5 4 ERS OEE eA EEOTO ooo REE ELE 6 31 Ater PS ae pe eee ae 6 oh 9 9 RA 64 eh ey ah ene 6 35 Section 6
364. pplied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Running Plates Using the Automation Controller Software Running the Instrument Starting the After you have loaded all plate documents into the Plate Queue or saved them the Plate Queue SDS Enterprise Database and you have configured the Instrument Control options you can start the plate queue To begin the plate queue click Start Queue from the Plate Queue tab The Automation Controller Software does the following 1 The Zymark Twister Microplate Handler loads the first plate from Stack 1 and places it into plate tray for the 7900HT instrument 2 The fixed position bar code reader scans the bar code for the plate 3 The Automation Controller Software does one of the following e If linked to the SDS Enterprise Database the software queries the database for the plate document with the bar code of the associated plate When found the software automatically downloads the plate document If the software does not find a plate document with a matching bar code in the database the software searches the contents of the plate queue as explained below e If not linked to a database the software searches the plate queue for the plate document with the bar code of the associated plate If found the Automation Controller Software loads the plate document information from the file system for the run 4 The 7900HT instrument loads the instrument
365. press on the sample block to ensure a good connection 3 Tighten the thumbscrew from step 9 on page 7 9 to secure the sample block locking bar to the instrument chassis may be a 5 32 inch hex bolt 4 Using the 5 16 inch hex key turn the sample block locking bolt clockwise until it is flush with the locking bar 5 Again press on the right and left sides of the front surface of the sample block to ensure that it is seated securely 6 Replace the thermal cycler access cover a Fit the lip at the bottom of the access cover over the lower edge of the bay b Push the cover towards the instrument until it snaps into place IMPORTANT You must reinstall the thermal cycler access cover before powering on the instrument Failure to do so prevents the instrument from uploading the firmware from the computer and causes the SDS software to display an error 7 10 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Replacing the Sample Block 7 If using a Plate Handler replace the covers for the fixed position bar code reader and the underlying platform removed in step 6 on page 7 8 8 Plug Fixed position bar code reader and underlying platform covers in and power on the 7900HT instrument 9 Confirm the function of the installed sample block module a Start the Automation Controller Software b Select the Thermal Status tab Does the software Th
366. prostate cancer patients In Process Citation Cancer Epidemiol Biomarkers Prev 8 10 901 905 1999 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Bibliography 7 Shi M M S P Myrand M R Bleavins and F A de la Iglesia High throughput genotyping method for glutathione S transferase T1 and M1 gene deletions using TaqMan probes Res Commun Mol Pathol Pharmacol 103 3 15 1999 Bibliography 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Glossary sdd sdm Sds sdt AC AAC AR 5 nuclease assay ABI PRISM SDS Detector file sdd SDS 7900HT Multiple Plate Document sdm Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide The file extension of an ABI PRISM SDS Detector file for SDS software for the 7900HT instrument The file extension of a SDS 7900HT Multiple Plate Document sdm for SDS software for the 7900HT instrument The file extension of a SDS 7900HT Document sds for SDS software for the 7900HT instrument The file extension of aSDS 7900HT Template Document sdt for SDS software for the 7900HT instrument The difference between the threshold cycle of a sample assay and the threshold cycle of the corresponding endogenous reference AC C target Cr Cy endogenous control For a given cDNA target the difference between the average AC value of a targe
367. ps M E Moreno C Bare J M McCune and D Ganem 1999 Experimental Transmission of Kaposi s Sarcoma associated Herpesvirus KSHV HHV 8 to SCID hu Thy Liv Mice J Exp Med 190 1857 1868 Bibliography 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Dolken L F Schuler and G Dolken Quantitative detection of t 14 18 positive cells by real time quantitative PCR using fluorogenic probes BioTechniques 25 1998 1058 1064 Eads C A Danenberg K D Kawakami K Saltz L B Danenberg P V and Laird P W CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression Cancer Res 59 10 2302 2306 1999 Eads C A Danenberg K D Kawakami K Saltz L B Blake C Shibata D Danenberg P V and Laird P W MethyLight a high throughput assay to measure DNA methylation Nucleic Acids Res 2000 Apr 15 28 8 E32 Eder M Battmer K Kafert S Stucki A Ganser A and Hertenstein B Monitoring of BCR ABL expression using real time RT PCR in CML after bone marrow or peripheral blood stem cell transplantation Leukemia 13 9 1383 1389 1999 Fink L et al Real time quantitative RT PCR after laser assisted cell picking Nat Med 4 1998 1329 1333 Haugland R A Vesper S J and Wymer L J Quantitative measurement of stachybotrys chartarum conidia using real time detection of PCR products with the TaqMan TM
368. r the centrifuge to 15679 Bucket type control Scrolling control IMPORTANT Be sure to set the correct bucket type Setting the correct bucket type ensures that the maximum rotational speed stays within the manufacturer s specified limits 3 Using the front panel controls set the following operational parameters Parameter Value Up ramp rate 9 Down ramp rate 9 Rotational speed 1200 rom 331 x 9 Centrifugation time 1 min rom indicator Buttons 1 4 are T T eas nea noon B ooann buttons Up ramp Down ramp rom Time rate control rate control controls controls 4 Power off the centrifuge Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 13 Chapter 4 Operating the Instrument About the Sealer Sealer Components Laboratory Setup The microfluidic card sealer is required to isolate the wells of the Low Density Array after it is loaded with cDNA samples or controls and master mix The sealer uses a precision stylus assembly carriage to seal the main fluid distribution channels of the Low Density Array Handle Low Density Array Used to move the carriage Shown foil side up with fill reservoirs on the right which contains the stylus carriage moves from left to right during sealing in assembly the direction of the arrows etched into the base Insert plate Holds the Low Density Array during sealing Base Provides a stable p
369. r 1 Product Overview 7900HT Instrument Internal Components Automated Plate Handling System Heated Clamp The 7900HT instrument contains the hardware used for thermal cycling and detection of fluorescent chemistries see page 1 2 Figure 1 3 illustrates the major subcomponents of the instrument described in detail below N o7 lol Do not remove the cover to the Applied Biosystems 7900HT Fast Real Time PCR System Only a qualified Applied Biosystems service engineer may repair or adjust the internal components of the 7900HT instrument Failure to comply can result in serious injury and or damage to the instrument Laser CCD Optics camera System Reader R Heated clamp Automated plate handling system Sample block module Figure 1 3 Internal Components of the 7900HT Instrument Lg ANA PHYSICAL HAZARD Keep hands and loose clothing away from the instrument tray and door at all times during instrument operation The 7900HT instrument contains several internal components that can cause serious physical injury The 7900HT instrument features an automated plate handling system to provide easy loading and removal of plates from the instrument In combination with the automation module the plate handling system allows unattended operation of the instrument AS erence PHYSICAL INJURY HAZARD Hot Surface Use care when working around this area to avoid being burned by hot components The 7900HT instrume
370. r Guide 5 13 Chapter 5 Analyzing End Point Data Manually Calling Allele Types l 2 Select the Results tab In the Allelic Discrimination Plot zoom out until all crossmarks are visible in the plot a Click amp magnifying glass tool b Click the plot to zoom out c Click F lasso tool d Select all of the marks within the plot by clicking and dragging the pointer across all datapoints in the plot The software outlines all selected wells within the grid view e Examine the tray pane to confirm that all wells are selected If not all wells are selected repeat steps a through d until all wells are visible on the plot ft 23 415 6 7 8 9110111213 14 15 16 1718 1920 212223 BERBER RRR RRR RRR RRR BERR ERR RRR RRR RRR RRR MIKIKI DIII RII RI bIRI RI DIbIRIDIRIRIIRIRIRIDIRS N XID XIII RII RIKI IRI RI DIRK EI bIRIRIRIRId KIDIRIB IIIA BKIK DIDIK DIII IIKI III Select the sample cluster exhibiting amplification of the first probe Setup Instrument Results MME caitfaiciex A k eA Marker fey Legend Allele X Allele Y Allele XR E NTC X Undetermined Allele CYP 2C19 2 1 Allele X homozygotes Allele X CYP 2C19 2 2 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Calling and Scrutinizing Allelic Discrimination Data 4 Select Call gt Allele X Setup Instrument Results Marker CYP 2 Call A
371. r Guide 7 55 Chapter 7 Maintaining the Instrument 7 56 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Troubleshooting In This Chapter Troubleshooting Table 0 0 0 cece nee nee 8 2 Low Precision or Irreproducibility 0 0 0 0 0 0 0c ccc eee 8 5 Backpround KUNS gg 5nd ind ode 4e4b 44944640 644 e eRe Eee ee eRe Sao 8 9 eE oo ed ea oo oe Beas eG ee eee dee eee eka es 8 11 Real Time Runs Quantitative PCR and Dissociation Curves 8 12 End Point Runs Allelic Discrimination 0 0 0 0 000 000 eens 8 13 Software and 7900HT Instrument 4 650s eee eee eee ew eee eee cee even peewee 8 14 Zymark Twister Microplate Handler and Fixed Position Bar Code Reader 8 17 TagMan Low Density Alay 4 4356445445499 6 ro ukine ariii erriei 8 18 So Caepio ee o oo hh es 4 ee ee REFERTE REEERE REET ees 8 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 8 1 Chapter 8 Troubleshooting Troubleshooting Table Overview The following table is designed to help you troubleshoot most of the problems you may encounter while using the Applied Biosystems 7900HT Fast Real Time PCR System The information in the table is arranged by category as follows e Chemistry problems e Run problems e Instrument and Automation Accessory Problems Each category contains subcategories followed by a brief description of the symptoms you might encounter
372. r Use y 0 cu sickened sues par baaewaddbuwen seas 4 8 Preparing TaqMan Low Density Arrays for Use 0 0 0 0 cece eee 4 10 Section 4 2 Running an Individual Plate cee ecw cece ee eee 4 23 Savine the Plate Document 55 soe c eye aa 549 045 94404400440 0040284 0445K05 4 24 Running a Single Plate Using the SDS Software 005 4 25 Aner e ee ee ee eee ee ee ee es ee eee er ee 4 29 Section 4 3 Automated Operation ccc cece cece cece cece eceees 4 31 Operating the Software with an SDS Enterprise Database 4 32 Operating the Software without an SDS Enterprise Database 4 34 Running Plates Using the Automation Controller Software 4 4 os ae oh he oe oe Oe Ce ewe ee bees ee deen eeaes 4 44 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 1 Chapter 4 Operating the Instrument Notes for Database Users Database Alerts and Notifications Reason s For Change Dialog Box Electronic Signature Verification Dialog Box 4 2 If you are using an SDS Enterprise Database to store SDS plate documents sessions studies and data you may be required to perform additional tasks when creating and modifying plate documents as described in this chapter This section describes the types of actions you may need to perform depending on how your administrator has configured the database For more information on any of the feat
373. r needs e Teach you to use the hand held bar code reader e Provide you with timesaving techniques that will increase your effectiveness with the SDS software Accessing the The Sequence Detection Systems Software Online Help provides a version of this Online Tutorial tutorial If you prefer to follow the online tutorial open the online help 1 Ifnot already active start the SDS software as explained on page 2 7 2 Select Help gt SDS Online Help 3 Inthe SDS Online Help click Basic Skills Tutorial from the list of options 4 Follow the directions displayed on your screen 2 10 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Basic Software Skills Tutorial Notes for Database Users Database Alerts Ifyou are using an SDS Enterprise Database to store SDS plate documents sessions and Notifications studies and data you may be required to perform additional tasks when performing the lessons in this tutorial This section describes the types of actions you may need to perform depending on how your administrator has configured the database Reason s For The Reason s for Change dialog box appears only if the SDS Enterprise Database is Change Dialog configured for auditing The auditing system monitors the creation modification Box and deletion of the SDS data contained by the database If the Reasons for Change dialog box appears do one of the following e Select a description for the
374. r person s eyes xxii Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Workstation Safety Workstation Safety Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions Nexen MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks e Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse e Position the keyboard mouse and monitor to promote relaxed body and head postures Safety and Electromagnetic Compatibility EMC Standards U S and Canadian Safety Standards c UL us Canadian EMC Standard European Safety and EMC Standards CE Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide This section provides information on e U S and Canadian Safety Standards e Canadian EMC Standard e European Safety and EMC Standards e Australian EMC Standards This instrument has been tested to and compl
375. r plates See page 4 1 10 Analyze the run data see page 6 5 Steps 2 and 2 can be eliminated by importing the plate document setup information from a tab delimited text file See Importing Plate Document Setup Table Files on page A 2 for more information 3 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Workflow Overview Relative Quantification 1 Create a relative quantification plate document see page 3 8 Workflow 4 2 Apply detectors to the plate document a Create detectors for the relative quantification probes See page 3 9 b Copy the detectors to the plate document See page 3 11 v 3 Configure the plate document with detector tasks NTC and Unknown see page 3 14 4 Program the method for the relative quantification run See page 3 17 5 Choose from the following If running a single plate then continue to step 2 If running the first plate in a series of plates with identical assay configurations then save the plate document as a template see page 3 22 6 Create a plate document from the template created in step 2 see page 3 24 7 Configure the document with sample names and plate information see page 3 25 8 Prepare and run the relative quantification plate or plates see page 4 1 9 Analyze the run data see page 6 15
376. r signal normalization in all 5 nuclease assays dNTPs and optimized buffer components Stored in and thus retrievable from the database used by the SDS software Pure dye data is generated from the results of a pure dye run in which the SDS software collects spectral data from a set of dye standards during a 2 min hold at 60 C The software stores the spectral information for the pure dye standards within a calibration file located in the SDS directory This calibration file is used for calculating multi component data After the run the software extracts each component dye spectrum from the collected data in the pure spectra run file Because the age and use of the instrument components can affect the pure spectra readings Applied Biosystems recommends updating the pure spectra data files once or twice annually depending on instrument use Glossary 5 quality value quantity quantity mean quantity standard deviation quencher dye ramp temperature ramp raw data reaction device real time run analysis relative quantification reporter dye Glossary 6 The quality value for a given point is an estimate of the probability that the assigned genotype for that point is correct with respect to the rest of the points in the data set The term quality value is only used for allelic discrimination autocalling 1 the quantity values for the standards contained on a plate 2 the initial sample quantity computed
377. r the SDS software Applied Biosystems recommends the following settings e End point runs allelic discrimination Xms20m Xmx60m e Real time runs absolute or relative quantifiation xXms80m Xmx90m Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 19 Appendix A Software Reference Using the Search Tool Programming The Search tool helps to organize the large set data in the Add files dialog box The Filters Search tool allows the software to display a subset of the total number of plate documents based on a set of predefined criteria The Search tool operates on a logic similar to a simple computer programming language Each filter consists of a single logical statement that instructs the software to include or exclude plate documents based on a specific parameter or quality The statements can be concatenated to screen the displayed data based on multiple criteria Search The Search tool consists of a table in which each row represents a single statement Statements _ that tests the associated data set for a condition Every time a row or statement is added to the table the software evaluates each plate document in the selected directory for the condition defined by the statement If a plate document meets the criteria defined by the test the test evaluates true then the software displays it in the Search Results list If a plate document does not meet the criteria the test evaluates
378. r which they are created Table 1 6 lists the average sizes of typical files produced by the SDS software Table 1 6 Average Plate Document File Sizes File Size Run Type Application Uncompressed Compressed Plate Read Allelic Discrimination 150 to 180 KB 70 to 90 KB Real Time Absolute Quantification 15 to 25 MB 10 to 15 MB Relative Quantification Compressed file sizes are estimates based on standard compression using the WinZip utility For more information see Archiving SDS Files on page 7 54 tThe maximum file sizes displayed above are nominal for real time runs absolute or relative quantification File size can increase depending on the plate document s data collection options tThe file size for a real time run depends on the length of the run and the configuration of the data collection options in the plate document Longer runs and runs configured to collect data at multiple stages of the method can be considerably larger than the 15 to 25 MB average IMPORTANT Because of memory constraints the SDS Enterprise Database cannot save real time plate documents greater than 40 MB Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Managing Sequence Detection System Data Managing Sequence Detection System Data System Operation and Dataflow Standard Modes of Operation Data management strategy is a crucial element of suc
379. rations Note Applied Biosystems protocols are available on the Applied Biosystems Company Web Site See How to Obtain Services and Support on page xii for more information Run Your Custom Note If conducting a quantitative PCR experiment consider the use of replicate Assay assays to enhance the precision of you data Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide B 3 Appendix B Designing TaqMan Reagent Based Assays Design Tips for Allelic Discrimination Assays Discrimination by Multiple Probes TaqMan Probe Design Guidelines By using different reporter dyes cleavage of multiple probes can be detected in a single PCR One application of this multi probe capability is to use allele specific probes to distinguish genetic polymorphisms Bloch 1991 Lee et al 1993 Probes that differ by as little as a single nucleotide will exhibit allele specific cleavage This is true even for probes with a reporter on the 5 end and the non fluorescent quencher on the 3 end Bloch 1991 IMPORTANT When designing probes it is important to consider probes from both strands Follow the guidelines in the table below for designing TaqMan MGB probes Table B 2 Guidelines for Designing TaqMan MGB Probes for Allelic Discrimination Priority Guideline Avoid probes with a guanine residue at the 5 end of the probe 1 A guanine residue adjacent to the reporter dye will quench the r
380. rd party program can be used to construct setup table files which can then be imported into plate documents for use To guarantee a successful incorporation of setup information from a text file to the plate document the file must e Be saved in a tab delimited text format e Conform to the setup table file formats described on page A 15 Importation of setup table data into a plate document is accomplished in three major steps Creating an Empty Setup Table File The first step in the procedure is to export a setup table file from a blank plate document Note The blank setup table file can be created using a secondary application such as Microsoft Excel or a text editor so long as it is saved in tab delimited format and is configured according to the file structure explained on page A 15 1 Start the SDS software 2 Click or select File gt New 3 Configure the New Document dialog box with the correct assay type and plate format for your experiment and click 4 Click or select File gt Export GN In the Look In field of the Export dialog box navigate to the directory you would like to receive the exported file Select Export gt Setup Table Select the All Wells radio button Select the SDS 2 2 1 radio button Se SS Click the File name text box and enter a name for the file 10 Click Beet The software exports the setup table data for the empty plate document as a tab delimited text fil
381. rd shortcuts using 2 28 L launching Automation Controller Software 4 39 SDS software 2 7 LAVA software about 1 14 aligning the fixed position bar code reader 7 49 to 7 51 launching 7 49 LDHost software See LAVA software learning to use the SDS software 2 10 lights See instrument status lights limited warranty statement E 1 Linear Phase of the amplification curve D 5 loading plates into the instrument 4 25 into the Plate Handler stacks 4 42 low copy templates 8 8 M maintenance schedule 7 3 managing SDS data 1 17 markers about 3 12 applying to a plate document 3 13 copying toa plate document 3 13 creating 3 12 importing into a plate document A 2 master mixes preparing B 3 using 8 6 maximizing instrument throughput 3 3 maximizing minimizing panes views and plots 2 22 maximum gene expression level 6 33 melting temperature definition 6 43 determining 6 44 methods about 3 17 to 3 21 adding a hold cycle set or step 3 19 adding a temperature ramp 3 21 Index 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide adjusting step parameters 3 19 configuring data collection options 3 20 programming 3 18 to 3 21 removing a step 3 19 setting the sample volume 3 20 minimum gene expression level 6 33 modes of operation 1 17 automated 1 18 database 1 18 stand alone 1 17 mRNA targets number per well 4 10 N no template control NTC detector task 3 14 NTC call
382. re continue to step 3 Otherwise create a plate document to receive the setup table data a Start the SDS software b Create or open a plate document to receive the information from the text file Click or select File gt Import In the Look In field of the Import dialog box navigate to and select the completed tab delimited setup table file from step 4 in the previous procedure Click Import The software imports the setup table information from the text file and automatically configures the plate document plate grid and setup table with detector detector task marker and sample data Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 3 Appendix A Software Reference Setup Table Files About the Setup Table File Format Format Overview Conventions The SDS software features the ability to import setup table information into a plate document from a tab delimited text file To guarantee a successful importation of setup table data the imported file must follow the setup table format outlined in this section The information in the setup table file must be ordered according to the following convention 1 File Version see page A 5 Plate Characteristics 2 Plate Size see page A 5 3 Plate ID see page A 5 Detector Definitions 4 Number of Detectors see page A 6 5 Detectors List Header see page A 6 6 Detectors List see page A 7 Marker Defini
383. re available after the analysis Changing the Display Settings 0 0 see below PO AICI ot cee edn ccd eeowam edhe Catone EE see below Exporting Plate Document Data 045 45 0 64iedeueeeaeeaad ieee eede see below Saving the Res lis ee ee ee ee 5 19 Before printing or exporting the analyzed data you can reconfigure the appearance of several elements of the plate document including the results table plate grid and most plots Allelic Discrimination Raw Data and Background plots 1 Click l or select View gt Display Settings 2 Inthe Display Settings dialog box click 7 for further instructions on modifying the display settings The SDS software can print a report of the analyzed data containing individual or multiple elements of the plate document 1 Click amp or select File gt Print Report 2 In the Print Report dialog box click the 7 for instructions on setting up previewing and printing the report Exporting Plate Document Data as a Tab Delimited Text File The SDS software can export raw or analyzed data in tab delimited txt format for all or a select group of wells on a plate document The exported files are compatible with most spreadsheet applications and programs that can read tab delimited text To export run data as a tab delimited text file choose one of the following for further instructions e See Exporting Plate Document Data on page A 16 e Click 7 within the table view
384. readsheet applications and programs that can read tab delimited text To export run data as a tab delimited text file choose one of the following for further instructions e See Exporting Plate Document Data on page A 16 e Click 7 within the table view Exporting Plots as Graphics The SDS software can export most panes and plots of the plate document as JPEG Joint Photographic Experts Group graphic files The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software To export a plot as a graphic file see Exporting Graphics on page A 16 or click m in the plot of interest for further instructions Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 51 Chapter 6 Analyzing Real Time Data Saving the The SDS software saves the results of the analysis differently depending on whether Results the analysis is saved to the SDS Enterprise Database see below or to a plate document file see page 6 54 Saving the Analysis Session to the SDS Enterprise Database If using the SDS Enterprise Database you can save your analysis of the run data to the database as an analysis session IMPORTANT Observe the following when saving sessions to the database e If you modified the plate document information in any way by removing wells from use or by changing the displ
385. reasingly smaller regions of the plate document see below ee a a ere es 8 E 3 E ca 3 a Al m E E E E E B B2 B3 B4 B5 B6 E E E E E fc c2 c3 c4 c5 c6 E E E E E D fo D2 D3 D4 D5 D6 E E E E E fer E2 E3 E4 E5 E6 E E E E E F ira F2 F3 F4 F5 F6 E E E E E G fo1 62 63 64 65 66 ae x T x E x a x a 8 E E ca E 3 a Ag D N D oO E 5 m oD BS B9 SH 3 a s s E C fc cs ca E E E E E D fo4 D2 D3 D4 D5 DB Ds pa E E E E E E fer E2 E3 E4 E5 E6 E E E E F fra F2 F3 F4 F5 F6 F7 Fa F9 E E E E E G foa 62 63 64 65 66 67 68 ca E 3 3 E a Ag 3H 3H m 5 a 3 EE 3 2 E E E E E E E E E c c2 c3 c4 c5 c6 c7 cs ca E E E E E E E8 E D D2 D3 D4 D5 DB D7 DS pa E E E E E E a E E E2 E3 E4 E5 E6 E7 ES E9 E E E E E a F F2 F3 F4 F5 F6 F7 Fs F9 E E E E E E E G fot 62 63 64 65 66 oF 68 69 Si ols x N x wo x B x a x a A10 A11 B10 c10 11 D10 D11 B11 E10 E11 F10 F11 10 o11 H10 H11 A10 A11 B10 B11 c10 11 D10 D11 F10 F11 610 o11 B10 B11 E E c10 11 E E D10 D11 A12 B12 C12 D12 E12 F12 G12 H12 A12 B12 C12 D12 F12 G12 Raw Data Calibration Data Amplitude
386. representation of a specific consumable that is a reaction plate or Low Density Array containing samples and reagents for use on the 7900HT instrument Plate documents contain the following information e Detector information and arrangement on the plate e Marker information and arrangement on the plate allelic discrimination only e Sample information and arrangement on the plate e Method parameters for the run absolute and relative quantification pd If not already open start the SDS software as explained on page 3 7 Click 4 or select File gt New 3 Configure the New Document dialog box with settings for the run e Assay drop down list Select the type of assay appropriate for your plate e Container drop down list Select the type of consumable you intend to run e Template drop down list Select Blank Template e Barcode field Do one of the following Creating a Plate Document If you are creating a Plate document to run a single plate scan or enter the bar code for the plate Plate Document Template for creating multiple plates leave the field blank IMPORTANT The SDS Enterprise Database does not support the creation of two plate documents of different run types with the same bar code Note If performing a dissociation curve experiment select Absolute Quantification from the Assay drop down list Assay Absolute Quantification Assay drop down list Container 384 Wells Clear
387. rescence After the Analysis User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to save analyzed data from a dissociation curve experiment Post Analysis Changing the Display Settings 20 nuan eee eee 6 52 Options Printing a Report 0 eee eee nee e rara 6 52 Exporting Plate Document Data 0 0 0 0000 ccc eee 6 52 Savna TS co cee Senne ea eben epee or aetetaghovedes oe ade 6 53 6 44 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 6 4 Procedure Reference Section 6 4 Procedure Reference In This Section Setting the Baseline and Threshold Values 0 0 0000 cece eeeeeeee 6 46 Eliminating Outlying Amplification 0 0 6 49 ear i ta yk ogy oe ee ae ee eee 0E de 5 bose en 6 52 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 45 Chapter 6 Analyzing Real Time Data Setting the Baseline and Threshold Values Setting the Ifyou choose to set the baseline and threshold values manually for any detector in the Baseline and _ study you need to perform the following procedure for each detector Threshold Values 1 Select the Results tab for the Run 2 If setting the baseline and threshold for a Absolute Quantification Plate Document Select all wells of the plate by clicking the upper left corner of the plate grid
388. reservoirs to its wells are open and undamaged 4 14 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Preparing TaqMan Low Density Arrays for Use Loading the Fill 1 When the packaging has reached room temperature and you are ready to load Reservoirs with PCR reaction mix carefully remove a Low Density Array from its packaging Sample Specific PCR Reaction Mix 3 Load 100 uL of the desired sample specific PCR reaction mix into a 100 uL micropipette Place the Low Density Array on a lab bench with the foil side down 4 Hold the micropipette in an angled position and place the tip in the fill port Note There is a fill port on the left arm of each fill reservoir it is the larger of the two holes Fill port AD Vent port TT I IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port m __ o 5 Dispense the sample specific PCR reaction mix so that it sweeps in and around the fill reservoir toward the vent port IMPORTANT Pipette the entire 100 uL into the fill reservoir IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port IMPORTANT Be careful when pushing the micropipette plunger to its second stop position to expel the sample specific PCR reaction mix from the tip Ifa large amount of air is released it can push the reaction mix out of the fill reservoir
389. retina and leave permanent blind spots Never look into the laser beam Remove jewelry and anything else that can reflect the beam into your eyes Protect others from exposure to the beam IMPORTANT If using the SDS Enterprise Database the bar codes entered must be unique and cannot be used by an existing plate document c Repeat step b for every plate in the batch d When finished click ok The plate bar codes appear inside the Plate ID field 5 Select a destination directory to store the new plate documents a Click Browse b In the Look in field navigate to and select the directory you want to use to receive the new files c Click Select directory i The Template Batch dialog box displays the selected destination directory in the Plate Directory field xl peal Plate ID 384N0257M0 a 384N00LQQC 384NO00LR9xX 384N00LQRD 384N025075 384NOOLON 384N016N9 E Import Clear Plate Directory Browse a Create Done 6 Click Create f The software creates plate documents for all entries listed in the Plate ID list saves them to the destination directory adds them to the plate queue and displays a message indicating the number of plate documents it created and sent to the plate queue aon amp wh New Destination directory 7 Click ox to close the message box 8 Click _ pone to close the Template Batch dialog box 9 Select File gt Close to close th
390. rise Database 4 32 Operating the Software without an SDS Enterprise Database 4 34 Operating the Software without an SDS Enterprise Database 4 39 Alerte Ps is 0 940 be ea hee ho he oe Hoes REEE EIEE Aaa aS 4 44 Pre Run The following tasks must be complete to run plates on the 7900HT instrument See Checklist the associated page number for details on each procedure Done Check See Page A background run has been performed in the last week 7 16 A pure dye run has been performed in the last 6 months 7 20 The instrument tray does not contain a plate 4 44 IMPORTANT The instrument tray must be empty to begin a run If the instrument tray contains a plate you must eject and remove it The output stack does not contain plates Overview The Applied Biosystems 7900HT Fast Real Time PCR System features the capacity for high throughput unattended operation through the use of the Automation Controller Software The Automation Controller Software coordinates the function of the 7900HT instrument the fixed position bar code reader and the Zymark Twister Microplate Handler to run batches of prepared plates with minimal user intervention The Automation Controller Software can run plates using plate documents stored locally on the computer attached to the instrument or across a network on an SDS Enterprise Database Options for Au
391. roduce a noticeable rise in fluorescent signal during the entire PCR the fluorescent signal produced by the well never crosses the threshold defined for the associated detector e Quantity For wells containing Unknown samples this column reports the extrapolated quantity value of the sample in the same units of measure as the standard Standard samples this column displays the quantity assigned to the well Note See page 3 15 for information on applying quantities to standard wells of the plate document The following two columns contain data only if a well is run as part of a replicate group e Qty Mean The arithmetic mean for the quantity values of the replicate group associated with the well e Qty stddev The standard deviation of the quantity values of the replicate group associated with the well e Status Indicates the analysis status of the sample data After the Analysis User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to save the analyze data of an absolute quantification run Post Analysis Changing the Display Settings 0 0 c eee eee eee 6 52 Options Printing a Report 0 cece ccc ence e cence eeeeeneeeeees 6 52 Exporting Plate Document Data 1 0 4 4 00 40ded eeeeed eebeeee eed ee 6 52 Saving tbe KOSS cn h0c tends stand 4p eeeddbeee ee beans Seek Gb eR eee RS 6 53 6 14 Applied Biosystems 7
392. roduced by the wells in the dilution series 4 Select Analysis gt Analyze The software analyzes the raw run data 5 Click H Show Raw Data Plot 6 In the Raw Data Plot determine the highest concentration of dye that does not produce a saturated signal and record it for future use Note Saturated signals are characterized by their high peaks that rise beyond detectable levels gt 65 000 fluorescent units and appear as plateaus on the Raw Data plot The concentration of the custom dye that yields the highest possible signal but does not saturate is the maximum concentration for use with the 7900HT instrument 7 Repeat steps through 6 for any additional custom dyes Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 27 Chapter 7 Maintaining the Instrument 8 Create a pure dye plate for the custom dye s by pipetting each custom dye to at least three columns of an optical plate at the concentrations determined in step 7 O 0 000 O 0 00 0 lan oa O O O O O J 2 J 5 D O O O O O O O O O C Custom Dye 8 D CY Q gt gt a E O O WL O O GOO 9 919100109109 O O OJOJOO OR OOOOC Custom Dye 5 gt D O O O O O O GR2107 IMPORTANT The optical configuration of the 7900HT instrument requires that each pure dye occupy at least three columns of the Pure Dye plate to permit adequate data col
393. rs Note The software automatically populates the Session Name field with the name of the associated plate document 3 Optional In the Add to Study field click then select an existing study or create a new study by clicking w and doing the following a Inthe Name field of the Configure the Create New Study dialog box enter a name for the study up to 128 characters The Creator field is not editable and displays your user name b In the Description field enter a brief description of the study up to 255 characters c Click d Click ox Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 5 19 Chapter 5 Analyzing End Point Data 4 In the Save Results to Database dialog box click x 5 In the confirmation dialog box click Saving the SDS 7900HT Document Although the software save any changes made to the appearance of a plate document and to the analysis settings it does not save the calls made during the analysis 1 Inthe SDS software select File gt Save As 2 Inthe Look in field of the Save As dialog box navigate to and select a directory for the software to receive the new file 3 In the File name field do one of the following e Enter a file name for the plate document file or e Enter or scan the bar code number for the plate into the field Note The SDS software does not require that the file name match the bar code of the corres
394. rse transcription step e Absolute quantities of the standard must be known by some independent means Plasmid DNA or in vitro transcribed RNA are commonly used to prepare absolute standards Concentration is measured by A and converted to the number of copies using the molecular weight of the DNA or RNA e Consider the stability of the diluted standards especially for RNA Divide diluted standards into small aliquots store at 80 C and thaw only once before use An example of the effort required to generate trustworthy standards 1s provided by Collins et al Anal Biochem 226 120 129 1995 who reported on the steps they used in developing an absolute RNA standard for viral quantification e Pipetting must be accurate because the standards must be diluted over several orders of magnitude Plasmid DNA or in vitro transcribed RNA must be concentrated in order to measure an accurate A value The concentrated DNA or RNA must then be diluted 10 10 fold to be at a concentration similar to the target in biological samples Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide B 5 Appendix B Designing TaqMan Reagent Based Assays B 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Kits Reagents and Consumables In This Appendix Interchangeable Sample Block Modules and Accessories 4 C 2 Consumables and Disposables
395. rter Quencher Description Comments CYP 2C9 2 1 FAM PDAR CYP 2C9 2 Al1 Example Probe CYP 2C9 2 2 VIC PDAR CYP 2C9 2 Al2 Example Probe xxx Number of Markers 1 Marker Allelex Alleley Description Comments CYP 2C9 2 CYP 2C9 2 2 CYP 2C9 2 2 PDAR CYP 2C9 2 Example marker Well Sample Name Detector Task Quantity Detector Task Quantity 1 Sample 1 2 Sample 2 3 z 384 Sample 48 Example Code A 9 Absolute quantification setup table files SDS Setup File Version 3 Output Plate Size 96 Output Plate ID 96JG729FF903 xxx Number of Detectors 1 Detector Reporter Quencher Description Comments RNase P FAM TAMRA RNase P Probe Example Probe Well Sample Name Detector Task Quantity 1 Sample 1 GAPDH UNKN 0 2 Sample 2 GAPDH STND 20000 3 54 96 Sample 30 GAPDH UNKN 0 Example Code A 10 Relative quantification setup table files xxx SDS Setup File Version 3 Output Plate Size 96 Output Plate ID 96FRE505SDA2 xxx Number of Detectors 2 Detector Reporter Quencher Description Comments GAPDH VIC GAPDH Probe Example Probe TNF Q FAM TNF a Probe Example Probe Well Sample Name Detector Task Quantity Detector Task Quantity 1 Calibrator GAPDH ENDO 0 TNF Q UNKN 0 2 Sample 1 GAPDH ENDO 0 TNF Q UNKN 0 3 T 96 Sample 12 GAPDH ENDO 0 TNF Q UNKN 0 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 11 Appendix A Software Reference Well Marker Information Element numbers 12 and 13 define the contents of the wells on
396. rter dyes If not the lack of reporter fluorescence may be caused by an absence of probe in the well a pipetting error e Background fluorescence Do all dyes fluoresce above the background The Background signal is a measure of ambient fluorescence If a dye fails to fluoresce above the background it is a strong indication that the well is missing probes labeled with the dye well does not contain probe PCR master mix or both e MSE Level The MSE mean squared error is a mathematical representation of how accurately the multicomponented data fits the raw data The higher the MSE value the greater the deviation between the multicomponented data and the raw data 8 12 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide End Point Runs Allelic Discrimination Amplification Plot The Amplification plot displays data from real time runs after signal normalization and Multicomponent analysis It contains the tools for setting the baseline and threshold cycle Cy values for the run What to look for e Correct baseline and threshold settings Are the baseline and threshold values set correctly Identify the components of the amplification curve and set the baseline so that the amplification curve growth begins at a cycle number that is greater than the highest baseline number IMPORTANT Do not adjust the default baseline if the amplification curve growth begins after cycle 15 Id
397. rument each plate must be sealed with the optical adhesive covers recommended above Applied Biosystems optical adhesive covers are specifically designed to permit the transmission of light to and from the wells of the optical plate IMPORTANT Do not use MicroAmp Optical Caps or MicroAmp Optical Tubes with the 7900HT instrument The instrument is not designed to run MicroAmp consumables which may damage its internal components if used Table C 2 Consumables and Disposables for the 7900HT Instrument Part No Description Quantity Optical Adhesive Covers 4313663 ABI PRISM Optical Adhesive Cover Starter Kit 20 Covers Includes 20 ABI PRISM Optical Adhesive Covers an Applicator and an ABI PRism Optical Cover Compression Pad 4311971 ABI PRism Optical Adhesive Covers 100 Covers 4360954 Optical Adhesive Covers 25 Covers 4323032 ABI PRISM Optical Caps 8 Caps Strip 300 Strips Pkg 2400 Caps Pkg 384 Well Optical Reaction Plates 4309849 ABI PRISM 384 Well Clear Optical Reaction Plate with 50 Plates Barcode code 128 4326270 ABI PRISM 384 Well Clear Optical Reaction Plate with 500 Plates Barcode code 128 10 Pack Includes 10 of PN 4309849 ABI PRiSm 384 Well Clear Optical Reaction Plates with Barcode Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide C 3 Appendix C Kits Reagents and Consumables C 4 Table C 2 C
398. rument while the lower side panel of the instrument is removed Doing so prevents the instrument from uploading the firmware from the computer and causes the software to display an error l 2 Power on the monitor and computer If using an automation module power on the Zymark Twister Microplate Handler Power HI POT A Ce ofeg eseo oes Rear panel of the Twister Power on the 7900HT instrument Power button Start the SDS software select start gt amp Programs gt Applied Biosystems gt amp SDS 2 2 1 gt amp SDS 2 2 1 If using the SDS Enterprise Database enter your user name and password in the appropriate fields of the Login dialog box then click ox TT xi User Mame Password Database Setup Cancel The SDS software displays the program window Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 7 Chapter 3 Preparing a Run Step 1 Creating a Plate Document User Access _ If using the SDS Enterprise Database you must belong to the Scientist or Requirement Administrator User Group to create plate documents About ABI PRISM Every plate run on the 7900HT instrument requires the creation of a plate document Plate Documents within the SDS software Each plate document is a virtual
399. rve for quantification of unknown Standards for samples you must apply the Standard task and quantity values to your absolute Absolute quantification plate document Quantification 1 In the plate grid of the SDS software select the replicate wells containing the first standard in the dilution series 2 In the well inspector click the field in the Task column for the appropriate detector entry and select the Standard from the drop down list 3 In the well inspector click the field in the Quantity column for the appropriate detector enter a quantity or concentration for the standard in the appropriate unit of measurement starting copy number picograms nanograms etc and press Enter The software labels the selected standard wells with the specified quantity os Selected wells E quantity applied to selection Quantity field 4 Repeat steps 1 through 3 to configure the plate with the other sets of replicate standard wells on the plate When finished the plate grid should contain a complete set of replicate wells labeled with the Standard task and assigned quantities that the software will use to compute the standard curve for the run 5 Do one of the following e Ifnecessary set the passive reference for the plate document as explained on Step 4 Setting the Passive Reference and Omitting Wells on page 3 16 e Otherwise program the method for your run as explained in Step 5 Programm
400. s see page B 2 Design the TaqMan probes and the forward and reverse primers see page B 2 Order reagents Quantitate the concentrations of the probes and primers see page B 3 Prepare the master mix see page B 3 Optimize the primer concentrations see page B 3 Run the assay see page B 4 Identify Target A target template is a DNA cDNA RNA or plasmid containing the nucleotide Sequence s sequence of interest For optimal results the target template should meet the following requirements The target nucleotide sequence must contain binding sites for both primers forward and reverse and the fluorogenic probe Short amplicons work best Amplicons ranging from 50 150 bp typically yield the most consistent results If designing assays for quantitative PCR see Design Tips for Quantitative PCR Assays on page B 6 for additional recommendations Design Probes The following sections contain general guidelines for designing primers and probes and Primers For specific design tips refer to the appropriate section for Allelic Discrimination see page B 5 and for Quantitative PCR see page B 6 Design Probe s for the Assay Adhere to the following guidelines when designing TaqMan probes Keep the G C content in the range of 30 80 Avoid runs of an identical nucleotide especially guanine where runs of four or more Gs should be avoided No Gon 5 end Keep the melting temperature T in the range of 68 70
401. s 4pplied Biosystems S S84N00LQ04 384N00LA00 S84N00L0 S84N00L02L 384N00LA3R S84N00LRA5 S84NO00LAD8 384NO008HKE 384N0257M0 384N00LQQC SB4NO0LAL lt 384N00LQRD S84N025075 S84N00LON 384N016N9 Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED QUEUED DONE DONE Instrument Control Robot s Active Stacks Open Close a Stack1 7 Stack2 I Stack3 7 dak I Restack when finished Plate stack check Studies boxes Save Allelic Discrimination to Study Example Study Save Relative Quantification to Study Ready 16 platefs Online 3 Ifyou want to retain the location of the plates in the stacks on the Plate Handler select the Restack when finished check box Restack when finished Instructs the arm to replace a stack of used plates to their original stack and in their original order after the stack has been run If the option is not selected the arm will place each group of used plates inside the next vacant stack in clockwise order beginning with the Output stack Note Re stacking plates adds significant operating time when running multiple plates Use the Restack when finished function only when necessary 4 Begin the plate queue as explained on page 4 43 4 42 A
402. s SNPs comprise the most common variants that are assayed using TaqMan reagents A PCR amplified section of DNA The Amplification plot displays data from real time PCR for gene expression assays after signal normalization and multi component analysis The Amplification plot appears only in plate documents containing analyzed data from real time runs It is a 2D plot of normalized reporter fluorescence for AR values versus the cycle number The AR versus cycle Amplification plot is used for calculation threshold cycle Cy A set of analysis results derived from a single raw data set associated with a single plate document analyzed by the SDS software The analysis session is persisted in the database and can be retrieved by the SDS software Multiple analysis sessions can be created from the same plate document however each analysis session must have a unique name An analysis session does not have to be associated with a Study however if it has such a relationship it can be associated with at most one Study at any given time Binding a probe to the DNA or cDNA during the PCR process A set of routines protocols and tools for building software applications A good API makes it easier for a programmer to develop a program by providing all the building blocks necessary to link the elements of the various subcomponents of the system With reference to the SDS 2 2 Enterprise Option software an assay type is one of the following alleli
403. s and Chemistries The Applied Biosystems 7900HT Fast Real Time PCR System is a second generation sequence detection system instrument designed for automated high throughput detection of fluorescent PCR related chemistries The instrument is capable of real time end point and dissociation curve analysis of assays arrayed on multiple formats The 7900HT instrument is optimized for use with Applied Biosystems chemistries for nucleic acid quantification and detection Figure 1 1 The Applied Biosystems 7900HT Fast Real Time PCR System with the Automation Accessory The Applied Biosystems 7900HT Fast Real Time PCR System provides two types of runs to support the variety of PCR related chemistries available from Applied Biosystems and affiliated companies End Point Plate Read Chemistry In an end point run the thermal cycling of prepared plates containing reagents and template is performed on a dedicated thermal cycler Following the PCR the 7900HT instrument is then used to collect a plate read or a single reading of the fluorescence resulting from the completed reactions Allelic Discrimination is an example of an end point chemistry that is directly supported by the SDS software Real Time Chemistry In a real time run the thermal cycling of prepared plates containing reagents and template is performed on the 7900HT instrument During the run the instrument collects data at each cycle of the PCR The resulting data provides a chronol
404. s does not contain the peak The contaminated well must be in the last four wells of columns 7 12 The raw data from the selected wells contains the peak The contaminated well must be in the last four wells of columns 10 12 The raw data from the selected wells contains the peak The contaminated well must be in the last two wells of columns 10 12 By selecting each of the wells from the last two wells of columns 10 12 the location of the contaminated well G10 is determined Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Pure Dye Runs 6 Repeat step 4 until you identify the location of each contaminated well 7 Decontaminate the sample block as explained in Decontaminating the Sample Block on page 7 14 8 Runa background plate to confirm that the contaminants have been removed If the contamination is present after running the background plate for a second time the background plate is likely to be the source of contamination Pure Dye Runs Pure Dye Table 8 3 Troubleshooting Pure Dye Runs Troubleshooting Table Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Observation Possible Cause Recommended Action Software will not extract pure dye data During plate setup the wrong plate type was assigned to the plate document Create and run a new pure dye plate document with the proper
405. s 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Basic Software Skills Tutorial 3 Select an entire column or row of wells using the headers a Click the header for row B to select all wells in the row The software outlines the wells of row B with a black border b Press and hold either the Shift or Ctrl key then click other columns or row headers to select multiple columns WEA k Ed vew Tools fount Aneyor Fandes iip aa 1112 3 4 5 6 7 8 9 toi 12 t3 141 Row B header sats Gee Ee SIDI AS aI III See e Teer EM it EE 4 Select all wells of the plate grid by clicking the top left corner of the plate grid The software outlines all of the wells in the plate document with a black border EE ai ER AmE sae 2 3 4 se 7 8 ios az esas Tie jE TT LELLELELELELELiLii LEEI BREE RRR RRR ERE EER EERE ERR RRR ERE EERE ERE REE EE BRR REE RRR SRR RRR RRR Ree Button i 5 Using the techniques illustrated in steps 1 to 4 practice selecting portions of the plate grid until you are comfortable using the feature Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 25 Chapter 2 Getting Started Exercise 3 You can zoom the plate grid to display the well information by clicking the Zoom Zooming the Grid button Plate Grid 1 Click Bl and observe how the grid expands to display the well information
406. s applying 5 15 NTC calls verifying 5 16 Number of targets per well 4 10 O L 6 33 opening instrument tray Automation Controller Software 4 44 instrument tray SDS software 4 29 plate documents 2 19 operating system supported 1 6 upgrading 7 55 operating the 7900HT instrument power switch 2 4 optimizing primer and probe concentrations B 3 outliers allelic discrimination 5 8 automatic outlier removal 6 19 outlying amplification multi reporter experiments 6 19 P panes hiding 2 21 maximizing minimizing 2 22 resizing 2 21 2 22 showing 2 21 passive reference setting 3 16 PCR 5 Nuclease Assay D 2 Geometric Exponential Phase D 4 kinetic analysis of D 4 Linear Phase D 5 Plateau Phase D 5 SYBR Green Chemistry D 3 PCR step fill reservoirs checking after centrifuging 4 18 fill reservoirs trimming after sealing 4 21 general practices 4 6 placing in bucket in centrifuge 4 17 TaqMan Low Density Array centrifuging 4 16 TaqMan Low Density Array loading 4 14 TaqMan Low Density Array placing in bucket 4 16 TaqMan Low Density Array sealing 4 19 persisted data analysis sessions 1 28 plate documents 1 28 studies 1 28 pipetting errors 8 6 pipettors using 8 6 plate adapter changing 7 12 plate document information applying to a plate document 3 25 plate documents 3 8 about 2 12 adding to the plate queue 4 35 4 40 applying detector tasks 3 14 applying sample names 3 25 assigning standard quantities 3 15
407. s explained in Step 3 Configuring the Plate Document with Tasks on page 3 14 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 3 11 Chapter 3 Preparing a Run Creating Markers Allelic discrimination plate documents feature the use of markers to aid in for Allelic organizing and applying detectors based on the loci they target A marker is a pairing Discrimination of two detectors representing chemical assays designed to discriminate between different alleles of a common locus The SDS software uses marker information during data analysis to organize and compare the processed run data IMPORTANT Allelic discrimination plate documents must contain at least one marker 1 Ifthe Detector Manager dialog box is open click _ bene to close it 2 Select Tools gt Marker Manager 3 In the Marker Manager dialog box click new 4 Inthe Enter name of new Marker field of the Add Marker dialog box enter a name for the new marker and click ox The new marker appears in the Markers field Note If using the SDS Enterprise Database you cannot create a marker that that uses the same combination of marker name and detectors as an existing marker 5 Apply detectors to the new marker a In the Markers field click the new marker to select it The software highlights the selected marker b In the Detectors list select the Use check boxes of the detectors that you want to assi
408. s line defines the total number of markers on the plate Number of Markers lt tab gt number of markers lt cr gt xxx Number of Markers 2 8 Markers List Header Allelic Discrimination Only IMPORTANT Marker List Header information is required only in setup table files created for allelic discrimination runs This section does not appear in setup table files created for absolute or relative quantification runs Description This line contains the column headings for the Marker Definitions section of the setup table file that make the file easier to edit using a program such as Microsoft Excel Format Marker lt tab gt AlleleX lt tab gt AlleleY lt tab gt Description lt tab gt Comments lt cr gt Example Example Code A 4 Marker Allelex AlleleY Description Comments A 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Setup Table Files 9 Markers List Allelic Discrimination Only IMPORTANT Marker List information is required only in setup table files created for allelic discrimination runs This section does not appear in setup table files created for absolute or relative quantification runs Description The marker list consists of one or more lines displaying the information for each marker used on the plate document e The Markers List section must contain one line or definition for each marker present on the plate e The number of lines in the Markers List
409. s of the analysis Saving data Progress bar saving the plate document or plate document template to a storage device The metered bar to the right of the message displays the progress of the action Importing data importing a file The metered bar to the right of the message displays the progress of the action Exporting data Progress bar exporting the data inside the current plate document to a file The metered bar to the right of the message displays the progress of the action About the The Instrument Status Icon indicates the status of the connection to the 7900HT Instrument instrument Status Icon The computer has successfully connected to the instrument and is awaiting a command Piviscomnected The computer is not able to connect to the instrument the instrument is not connected or is powered off Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 2 9 Chapter 2 Getting Started Basic Software Skills Tutorial User Access _ fusing a computer linked to an SDS Enterprise Database you must have Scientist or Requirement Administrator access privileges to complete this tutorial About This This tutorial will Tutorial e Teach you to create save print export and import SDS plate documents e Familiarize you with the basic components of the SDS software interface e Explain how to customize and arrange the user interface to suit you
410. s possible to the probe without overlapping the it e Use an annealing temperature of 58 60 C for quantitative PCR and 58 60 C for allelic discrimination except for TaqMan PDARs for Allelic Discrimination Quantitate the Use a spectrophotometric method to determine the concentrations of the probes and Probes and primers received See the JagMan Universal PCR Master Mix Protocol Primers PN 4304449 or the TaqMan Fast Universal PCR Master Mix 2X Protocol PN 4351891 for specific information about primer and probe quantification Prepare Refer to the TaqMan Universal PCR Master Mix Protocol PN 4304449 or the Master Mix TaqMan Fast Universal PCR Master Mix 2X Protocol PN 4351891 for specific information about preparing the master mix for use IMPORTANT PCR master mix used with the 7900HT instrument must contain a passive reference dye The SDS software uses the signal from the passive reference to normalize the reporter fluorescence making well to well comparisons possible All Applied Biosystems master mix products contain an optimal concentration of the ROX passive reference dye Note Applied Biosystems protocols are available on the Applied Biosystems Company Web Site See How to Obtain Services and Support on page xii for more information Optimize Refer to the ZaqgMan Universal PCR Master Mix Protocol PN 4304449 for Primer Probe specific information about optimizing primer probe concentrations Concent
411. s see een neee ay kae eey EET eE 7 30 Maintaining the Automation Accessory Adjusting the Sensitivity of the Plate Sensor Switch 00000 7 37 Allene tbe Plate Handler ss ncos on gccn edb badges Baur a ade aduwes i 7 41 Aligning the Fixed Position Bar Code Reader 0 0 00 cee eens 7 49 Cleaning and Replacing Gripper Finger Pads 0 0 e eee eee 7 52 Note The Automation Accessory includes the Zymark Twister Microplate Handler and the fixed position bar code reader See Instrument Components on page 1 3 for more information Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Getting Started Using the SDS The Sequence Detection Systems Software Online Help can guide you through the Software procedures for setting up performing and analyzing runs To get help at any time Online Help click 2 Help button located inside the dialog box or window in which you are working The SDS software provides two ways to access the online help To access general help get help for using a specific dialog box plot or feature Then select Help gt SDS Online Help click help button located inside the dialog box or window in which you are working For More For information about the Applied Biosystems 7900HT Fast Real Time PCR System Information or the SDS software Applied Biosystems recommends the following references e Appl
412. se Database allows two or more users to open and modify data simultaneously If another user has opened analyzed and saved the same data that you have just analyzed the software overwrites the analysis when you save yours to the database e Analysis session results are directly linked to the configuration of the plate document used to create the session If after you have saved your analysis session to the database you modify the linked plate document by omitting wells altering sample names etc your analysis session may be orphaned 1 e have no relationship to its attached plate document Consequently the results of your analysis session may change if you reanalyze it because you have changed the content of the associated plate document containing the raw data Applied Biosystems recommends the following guidelines for using analysis sessions Save the plate document as a new document any time you make changes to the plate setup omit wells alter sample names modify detectors or markers etc Maintain only one saved analysis session per document Understand that only results sessions saved after the latest changes to a plate setup are guaranteed to correspond to the linked plate document To save the results to the database 1 Select File gt Save Results to Database 2 Inthe Description field of the Save Results to Database dialog box enter a brief description of the plate document up to 255 characte
413. se User Guide Opening the Run Data Opening the Run Data User Access There is no access requirement All users can open allelic discrimination data that Requirement has been saved to the SDS Enterprise Database Opening the Opening a Plate Document File Data from a 1 Completed Run 2 3 4 Click or select File gt Open In the Look in field navigate to and select the plate document file Click Open i The SDS software displays the plate document file Configure the Analysis Settings for each marker as explained below Opening Un Analyzed Data from the SDS Enterprise Database l 2 Click or select File gt Open Document from Database In the Plate Query dialog box configure the Find Plates Matching These Criteria table with parameters that correspond to the document of interest Click Search E Plate Query Ea Find Plates Matching These Criteria variable Condition valei vaez o e e i 2 _ Funtime ae 12 13 PM ats anaes 12 13 PM Configure queries here Search Clear Row Clear All Append Query Results Click to begin Search Results the search SBano06SV2 BIE ST PH Alle Decrmiaton ERAT EP eqNO0Esv2 eje 02 11 04 AM Alec Discrimination DEFALLT H Documents that Se4NOOSSV2 6 6 02 9 98PM Relative Quarti Derat meet the search Open Select All Deselect All Clear All criteria appear here Done In the Search Results list scroll to and s
414. sed to lubricate the insides of plastic gloves often contains fluorescent compounds Use only powder free gloves and do not needlessly touch the reaction plates or optical adhesive seals Errors Human errors from time to time are inevitable such as pipetting into the wrong well or making a dilution mistake Human error can be reduced in the following ways e Perform the assay in a systematic fashion For example the pattern of sample positions should be simple avoid putting gaps in the rows e When pipetting the master mix look directly down into the reaction plate so that you can verify the transfer of the solution e If adding a small volume reagent such as template place the drop of liquid on the side of the well Briefly tap or centrifuge the plate afterwards to bring the droplet down into the well e After all pipetting is complete visually inspect all the wells to confirm the presence of the reagent drops Tapping or centrifuging the reaction plate will cause all the drops to slide down into the wells simultaneously e When making serial dilutions be sure to change the pipet tip after each dilution step e Visually inspect the liquid volumes being pipetted to verify that the volume is approximately correct A common mistake is using the wrong pipettor volume setting such as setting 20 uL instead of 2 0 uL e Visually inspect the volumes of the completed reactions looking for any wells that have volumes that do not matc
415. ser name and password then click x 2 After the software validates your user name and password it displays a message stating the success or failure of the signature Click to continue E Electronic Signature Verification fou are attempting to change for Property X of the curren document Description of the action that requires a signature Enter your user name Enter your password Figure 3 2 Electronic Signature Verification Dialog Box Options Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Before You Begin Before You Begin Background Information Getting More Information from the SDS Online Help Maximizing Throughput for End Point Runs Chapter 5 Analyzing End Point Data and Chapter 6 Analyzing Real Time Data include brief discussions of the experiments that you can perform using the 7900HT instrument Before beginning you may want to review the appropriate chapter for your experiment Allele LSE co cc ee ooh vba eneas bh bb eee Sheba ee beob4ae HER 5 5 Absolute Quantification 66 bch eee eee 4aGs ae Beas Pandaasereed bade 6 5 Reve Quantification o ee os bo bee noe bbe eae a beaded 6 15 Dissociation Curve Analysis 0 0 eens 6 37 The Sequence Detection Systems Software Online Help can guide you through the procedures for setting up performing and analyzing runs To get help at any time click the 2 button locate
416. sessions with studies uay vata However only users belonging to the Scientist or Administrator User Group can create a new study About the Study Associations The Automation Controller Software can automatically associate the data collected from plate runs called sessions with a specific study Attaching session data to studies is optional and can be done later if necessary using the RQ or SNP Manager Software See Database Design and Information Management on page 1 27 for more information about the use of studies IMPORTANT After you have attached a session to a study it cannot you cannot add it to another study until you have unattached it using the RQ or SNP Manager Software To specify the Study associations 1 In the Automation Controller Software dialog box do one of the following To set the study association for e Relative Quantification runs Click in the Save Relative Quantification to Study field or select Enterprise gt Select RQ Study e Allelic Discrimination runs Click in the Save Allelic Discrimination to Study field or select Enterprise gt Select AD Study Studies __Click to set the study association for relative quantification runs Click to set the study Ready 16 plateis Online association for allelic discrimination runs Save Allelic Discrimination to Study Example Study Save Relative Quantification to Study E 2 Inthe Select Study dialog box e
417. site at www appliedbiosystems com Applied Biosystems is committed to providing the world s leading technology and information for life scientists Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 06 2010 Applied Biosystems Part Number 4351684 Rev C
418. ssay Development Guidelines on page B 2 e The amplicon should span one or more introns to avoid amplification of the target gene in genomic DNA e The primer pair has to be specific to the target gene and does not amplify pseudogenes or other related genes e Test amplicons and select those that have the highest signal to noise ratio such as those yielding low Cys with cDNA and no amplification with no template control or genomic DNA e Ifno good sequence is found it may be necessary to examine the sequence and redesign the amplicon or simply screen for more sites If the gene you are studying does not have introns then you cannot design an amplicon that will amplify the mRNA sequence without amplifying the genomic sequence In this case it may be necessary to run RT minus controls Selecting and To ensure accurate results the standards used for absolute quantification must be Preparing carefully engineered validated and quantified before use Consider the following Standards for critical points for the proper use of absolute standard curves Absolute e The DNA or RNA used must be a single pure species For example plasmid Quantification DNA prepared from E coli often is contaminated with RNA which increases the A measurement and inflates the copy number determined for the plasmid e In general DNA cannot be used as a standard for absolute quantification of RNA because there is no control for the efficiency of the reve
419. stacks are used to hold the sample plates while the fifth is used by the Plate Handler to hold the first set of analyzed plates Maximally 20 plates can be placed in each stack The instrument can process maximally eighty plates in one batch A study in the SDS Enterprise Database option is a named collection of zero or more AD or RQ analysis sessions A study in the SDS software main application is an sdm plate document file that contains one or more sds relative quantification plate document files A setting applied to the detectors within a well of a plate document that determines how the data collected from the well during analysis are used The task selections available differ depending on the plate s assay type Glossary 7 template threshold threshold cycle C 1 SDS software SDS 7900HT Template Document or plate document template A pattern for creating an SDS 7900HT Document plate document The filename extension for a plate document template is sdt The plate document template facilitates creation of plate documents in the SDS software It is typically a starting point for batch document setup The binary representation of the plate document template is persisted in the database allowing the plate document template to be retrieved and then populated with data and saved as an SDS 7900HT Document 2 Chemical the DNA sample that is being amplified during PCR An arbitrary level of normalized reporter signal
420. standard B 10 O O Oi 5000 OOOO re sro a0 sr 2500 GR2108 The TaqMan Low Density 7900HT Installation Array TGF B card is an experiment run to verify the performance of the 7900HT instrument for use with TaqMan Low Density Arrays The TGF B card is preloaded with the reagents necessary for the detection and quantification of genomic copies of the TGF B gene Each well contains preloaded TGF B primers and FAM MGB dye labeled probe RNase P Runs e Appropriate TaqMan RNase P Instrument Verification Plate see Table 7 2 on page 7 30 e Centrifuge with plate adaptor TGF B Runs e TGF B card e TaqMan 2X Universal PCR Master Mix e Water DNase RNase free deionized e 2 mL microtube sterile e Centrifuge e Microfluidic card sealer Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 31 Chapter 7 Maintaining the Instrument Preparing the Preparing a TaqMan RNase P Instrument Verification Plate pl Fe 1 Remove the TaqMan RNase P Instrument Verification Plate from the freezer OF P Car and allow it to thaw to room temperature 2 Briefly centrifuge the TaqMan RNase P Instrument Verification Plate 3 Continue with Preparing the Plate Document on page 7 33 Preparing a TGF B Card 4 Remove the TGF B card from the refrigerator and allow it to warm to room temperature 5 Ina sterile 2 mL tube prepare the following PCR reaction mixture Reage
421. stem and SDS Enterprise Database User Guide Chapter 7 Maintaining the Instrument Notes for Database Users s c 02 oe wee Gee ee Eee ee ea eee 7 2 Recommended Maintenance Schedule 0 0 cee ees 7 3 Section 7 1 Maintaining the 7900HT Instrument 0 00 eee eee 7 5 Replacing the Sample BOCK x o 9 44te sd4 204450 be Ketek ew ee ee eee 7 6 Changing the Plate Adapter 0 ccc ete ees 7 12 Decontaminating the Sample Block 0 00 cece eee 7 14 Performing a Background Run 2 eee 7 16 Preparing the Background Plate or TaqMan Low Density Array 1 17 Creating a Plate Document for the Background Run 02 005 7 17 Running the Prepared Background Plate or TaqMan Low Density Array 7 18 Analyzing the Background Data 0 0 ccc eee 7 19 Performing a Pure Dye Run 25 eieacdst DvGud ose ete eee te FSO 4 Rae we ok 7 20 Preparing the Pure Dye Plates or Cards 0 00 cece ees 1 22 Preparing a Plate Document for a Pure Dye Plate or Card 1 22 Running the Prepared Pure Dye Plate or Card 0 0 0 0 eee ee ees 1 24 Analyzing the Pure Dye Run 1 eee 7 25 Adding Custom Dyes to the Pure Dye Set 0 00 cc eee 7 27 Verifying Instrument Performance 0000 cee eee ee eee 7 30 Section 7 2 Maintaining the Plate Handler 0 00 ees 7 35 Automation Accessory Components and Stack Positions 0
422. stems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide date stamps Cancel 3 9 Chapter 3 3 10 Preparing a Run 4 5 Add Detector Ea Name EMase P Group Applied Biosystems Assays Description Human RMase F Gene Assay Reporter F AN X Quencher Mon Flucrescent Color o Motes This 13 an example of a detector constructed for Ahsolute Relative T Ce ee TA Add Detector x Mame CYP 2C9 Allele 13 Group PDAR Description Pre Developed Assay and Reage Reporter F AK X Quencher Mon Flucrescent Color O Motes This is an example of a detector constructed for Allelic Discrimination Created Oct 24 2002 5 24 43 PM Last Modified Oct 24 2002 5 24 43 PM Last Modified Nov 3 2002 8 23 18 PM Cancel Cancel Allelic Discrimination Absolute or Relative Detector Quantification Detector Click The software saves the new detector and displays it in the detector list Created Nov 3 2002 6 23 18 PM Note If using the SDS Enterprise Database you cannot create a detector that that uses the same combination of detector name reporter dye and quencher dye as an existing detector Detector Manager Find D Fiter Fiter Settings Guencher Modification Date Creation Date Sun Nov 03 22 38 01 PST 2002 Sun Mov 03 22 38 01 PST 2002 B Sun Nov 03 22 38 25 PST 2002 Sun Mov
423. strument or e Select File gt Exit to close the SDS software 2 Start the Automation Controller Software select ig stat gt Programs gt amp Applied Biosystems gt amp SDS 2 2 1 gt F Automation Controller 2 2 3 Ifusing the SDS Enterprise Database enter your user name and password in the appropriate fields of the Automation Controller Log In dialog box then click 4 Ifusing the SDS Enterprise Database specify the Session Study associations as described in Associating Session and Study Data on page 4 33 5 Ifrunning the Automation Controller Software using the plate queue verify that Plate Queue field contains plate documents for all plates you intend to run e To add a plate document to the plate queue see Adding Plates to the Plate Queue on page 4 40 e To remove a plate document from the plate queue see Removing Plate Documents from the Plate Queue on page 4 40 Ii SDS Automation Controller mes Ea Fie Instrument Enterprise Help Plate Queue Thermal Status Run Type C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program FilesApplied Biosystems C Program Files Applied Biosystems S C Program Files Applied Biosystems C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program Files Appli
424. systems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 21 Chapter 4 Operating the Instrument 4 22 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Section 4 2 In This Section Pre Run Checklist Workflow Overview Section 4 2 Running an Individual Plate Running an Individual Plate Saving the Plate Document Running a Single Plate Using the SDS Software After the Run The following tasks must be complete to run plates on the 7900HT instrument See the associated page number for details on each procedure IMPORTANT The instrument tray must be empty to begin a run If the instrument tray contains a plate you must eject and remove it Done Check See Page A background run has been performed in the last week 7 16 A pure dye run has been performed in the last 6 months 7 20 The instrument tray does not contain a plate 4 44 1 Save the SDS Plate Document See page 4 24 2 Load and run the prepared optical plate or Low Density Array see page 4 25 3 Analyze the run data Allelic Discrimination Analysis See page 5 5 Absolute Quantification Analysis see page 6 5 Relative Quantification Analysis See page 6 15 Dissociation Curve Analysis see page 6 37 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Datab
425. t Example Code A 2 Allelic discrimination setup table file xxx SDS Setup File Version Output Plate Size Output Plate ID xxx Number of Detectors Detector Reporter CYP 2C9 2 1 FAM CYP 2C9 2 2 VIC 3 384 384N75822034 2 Quencher Description PDAR CYP 2C9 2 All PDAR CYP 2C9 2 Al2 Comments Example Probe Example Probe Example Code A 3 Absolute or relative quantification setup table file SDS Setup File Version eee QUEPUCY Plate Size Output Plate ID xxx Number of Detectors Detector Reporter GAPDH VIC SYBR Green SYBR RNase P FAM 3 96 960G729FF903 3 Quencher TAMRA Description GAPDH Probe SYBR Green I RNase P Probe Comments Example Probe Example Probe Example Probe Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 7 Appendix A Software Reference Marker Definitions Element numbers 7 to 9 define the markers that appear in the well descriptions that follow in a later section The marker definition consists of three sections the declaration of the number of markers the marker list header and the marker list 7 Number of Markers Allelic Discrimination Only Description Format Example IMPORTANT Number of Markers information is required only in setup table files created for allelic discrimination runs This section does not appear in setup table files created for absolute or relative quantification runs Thi
426. t sample and the average AC for the corresponding calibrator sample AAC test sample AvgAC test sample AvgAC calibrator sample This value is used to calculate expression fold value by the equation Expression fold value D aaCr The normalized reporter signal minus the baseline signal The baseline is established in the first few cycles of PCR Like R AR is not a fixed value but increases during PCR when the amplicon copy number increases until the reaction approaches a plateau The 5 five prime nuclease assay in combination with the TaqMan probes serves as the basis for real time polymerase chain reaction PCR technology on the 7900HT instrument See Fundamentals of the 5 Nuclease Assay on page D 2 for a detailed description of the reaction The SDS detector file is a tab delimited flat file that is used to transfer detector information to and from the SDS software using the Export and Import functions of the Detector Manager software The file contains a a header SDS2 2 1 GDF Global Detector File b tab delimited data for each detector allele Name the name of the reporter dye the name of the quencher dye a description a comment the owner the creation date and time the modification date and time and the color used to code the detector in graphical displays of the SDS software Multiple plate documents are used for the analysis of relative quantification data Glossary 1 SDS 7900HT D
427. t Vertical Home Click FindPlate The Plate Handler arm lowers onto the plate Save the rotary and vertical offset information d Click 1 Rotary Offset and click _tes The software records the rotary position for the plate drawer Zymark position 2 b Click Qvenicalortset and click Yes The software records the vertical position for the plate drawer Re checking the Now that the positions of the Plate Handler and instrument are fixed the Plate Input Stack 1 Handler stacks can be aligned and the positional values recorded l 2 Place an empty plate into input stack 1 In the Zymark Twister Software click the icon for stack 4 The Plate Handler arm moves over the input stack Using the Vertical Positioning commands lower the Plate Handler arm until it is cm above the stack and verify that it is centered on the stack If necessary center the stack using the Rotary Adjustment arrows Carefully lower the Plate Handler arm into the stack Center the gripper as it moves down the stack by adjusting the Rotary Adjustment arrows if needed After the Plate Handler arm is centered inside the stack click 4 Find Plae The Plate Handler arm lowers upon the plate Confirm the following e The plate is in the middle of the gripper span e The plate sensor switch is contacting the plate e The gripper does not contact the side of the stack Click Close Gripper Click Ti Yertical Home The
428. t plate there is a thumb slot to help you easily access one side of the Low Density Array Thumb slot 3 Inspect the Low Density Array for proper sealing The indentations from the stylus assembly should match up with the Low Density Array s main channels If the indentations do not match up or if the foil is in any way damaged do not use the Low Density Array 4 Return the carriage to its starting position on the base of the sealer IMPORTANT Do not move the carriage back before removing the Low Density Array Trimming Off the 1 Using scissors trim the fill reservoirs from the Low Density Array Use the edge Fill Reservoirs of the Low Density Array s carrier as a guide N OZON PHYSICAL INJURY HAZARD Take care when trimming off the fill reservoirs Use scissors rather than razor blades or other unprotected cutting devices The Low Density Array is now ready to be run on the 7900HT instrument 2 Choose from the following If performing Absolute or Relative Quantification or Dissociation Curve Analysis Run the plate as explained in Running the Plate on page 4 25 Allelic Discrimination a Load the plate onto a designated thermal cycler and perform the PCR b Briefly centrifuge the plate to draw the reactions to the bottom of the wells and to eliminate any air bubbles that may have formed during thermal cycling c Run the plate as explained in Running the Plate on page 4 25 Applied Bio
429. t template for a TaqMan RNase P Instrument Verification Plate The SDS software automatically installs several default plate document templates to AppliedBiosystems SDS2 2 1 Templates In addition to the RNase P Plate document template Applied Biosystems provides in the same directory several plate document templates for plates run regularly on the 7900HT instrument 1 Click gl or select File gt Open 2 In the Look In field of the Open dialog box navigate to AppliedBiosystems SDS2 2 1 Templates 3 Select File of type gt SDS 7900HT Template Document sdt 4 Inthe Look In field select the 96 Well RNaseP Install Plate sdt file Look in Templates T 384 well RNaseP Install Plate sdt T 96 Well Pure Dyes Plate 1 sdt T 96 Well Pure Dyes Plate 2 sdt i 96 Well RNaseP Install Plate sdt Select File name 96 Well RMaseP Install Plate sdt Open Files of type aer Prism 5D5 Template Document sdt Cancel 5 Click open The software opens the plate document template 6 Export the plate setup information from the plate document template as explained on page 2 15 2 14 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Basic Software Skills Tutorial Exporting the Plate Setup Information l na S i E di Click J or select File gt Export In the Look In field of the Export dialog box navigate to Applied Biosystems SDS Documents
430. t to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Chemical Safety Guidelines Chemical Waste Safety Read and understand the Material Safety Data Sheets MSDS provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page xviii Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Chemical Waste Safety Chemical Waste N OON HAZARDOUS WASTE Refer to Material Safety Data Sheets and Hazard local regulations for handling and disposal Nem CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can caus
431. tab In the Real Time tab of the Instrument tab click Spenjclose Place the background plate or Low Density Array into the instrument tray as shown below Well A1 Position the plate so that the bar code faces towards the front of the instrument Well A1 Position the Low Density Array so that the bar code faces towards the front of the instrument Note The A1 position is in the top left side of the instrument tray 7 18 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Performing a Background Run 2 Click start The 7900HT instrument begins the background run Note Before starting the run the instrument may pause up to 15 min to heat the heated cover to the appropriate temperature 3 When the background run is complete and the Run Complete dialog box appears a Click ox to close the dialog box b Click openjctose and remove the plate from the instrument tray c Extract the background component as explained on page 7 19 Analyzing the Background Data Extracting the In this procedure you will extract the calibration values from the background plate Background document After extraction the SDS software stores the data as part of the calibration file located in the Calibration subdirectory of the SDS directory 1 Select Analysis gt Extract Background The software attempts to extract the background signal and displays the success of the e
432. tabase you must belong to the Scientist or Administrator User Group to save a plate document as a plate document template Because plate documents created from the plate document template will retain its display settings configure the display settings of the plate document template as you would like the child plate documents to be displayed l 2 4 Click m select View gt Display Setting In the Display Settings dialog box configure the display settings for the E Results Grid and the Results Table Display Settings f x Select Pane view p Results rid Properties free j 1 Sa a eee Attribute H4 Results Table i ell Caley P Background Plot P Raw Data Plot Sample Allelic Discrimination Plot Marker Color Marker Call Number of Markers More Op More Down lt 1 lt 1 lt 1 511 a Use as defaults for Allelic Discrimination documents Apply Cancel For more information on the Display Settings dialog box or to view the procedures for configuring the display settings for the plate document template click 7 to open the Sequence Detection Systems Software Online Help When finished click The SDS software applies the new display settings to the plate document Go to Saving the Plate Document as a Template Optional on page 3 23 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Step 6 Saving the Plate Docum
433. tabase User Guide Database Design and Information Management SDS User The SDS User Account Manager Software installs with the version of the SDS Account Manager software that includes the database client software The software allows users of the Administrator User Group to assign privileges and passwords for user accounts and to add and remove them See the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide PN 4351669 for more information Database Design and Information Management About the The SDS Enterprise Database persists three primary types of retrievable data used to Study Centric operate the 7900HT instrument and to analyze the collected run data Design e Plate documents e Analysis sessions e Studies Figure 1 18 describes the sources and uses of the different types of data persisted to the database throughout the operation of the Applied Biosystems 7900HT Fast Real Time PCR System Prepared plates are sealed and loaded into the stacks of the Automation Accessory Just before each run the Plate documents are Automation Controller Software loads created on a client Plate Plate the plate and scans its bar code The computer using the SDS Documents Database Documents software then automatically downloads software and saved to the Server the plate document with the matching SDS Enterprise Database bar code from the database
434. tasks See detector tasks determining melting temperatures 6 44 diagram database LAN setup 1 23 database WAN setup 1 24 disconnecting the SDS software 4 30 display settings adjusting 3 22 dissociation curve about 6 38 analyzing 6 40 to 6 44 definitions of the T value 6 43 procedure checklist 6 22 6 40 programming atemperature ramp 3 21 Dissociation Plot about 6 42 exporting as a graphic file A 16 exporting data as a text file A 16 E ejecting a plate from the 7900HT instrument 4 29 4 44 endogenous control 6 20 end point runs about 5 2 allelic discrimination See allelic discrimination entering bar code information 2 27 3 25 4 36 comments 3 9 3 25 sample names into a plate document 3 25 Equipment needed 4 12 exportable data type definitions A 17 exporting data from a plate document 2 14 A 16 plots and views as graphic files A 16 External endogenous control assay 4 11 F finger pads cleaning 7 52 replacing 7 52 fixed position bar code reader aligning 7 49 to 7 51 connections 1 10 location 1 7 specification 1 7 fluorescent contamination 8 7 detection system 1 10 fluorogenic probe about D 2 designing B 2 to B 3 G gene expression level maximum 6 33 minimum 6 33 gene expression levels 6 31 Gene Expression Plot 6 31 Genomic DNA contamination minimizing 4 11 genotypic segregation of datapoints Allelic Discrimination Plot 5 8 Geometric Exponential Phase D 4 graph Gene Expression Profile sa
435. te to collect the reactions at the bottom of the wells and Visually verify that each reaction is positioned at the bottom of its well Correct Position Incorrect Position The sample is positioned correctly in the bottom of the well The sample lies on the side wall because the plate was not centrifuged An air bubble lies at the bottom of the well because the plate was not e Centrifuged with enough force or e Centrifuged for enough time 5 Ifrunning an ABI PRISM 96 Well Optical Reaction Plate apply the appropriate compression pad to the sealed optical plate If you are going to run the plate e Individually using the SDS software then apply a standard compression pad Figure 4 3 e As part of a batch using the Automation Controller Software then apply an ABI PRISM Snap On Compression Pad Figure 4 3 IMPORTANT The ABI PRISM Snap On Compression Pad plate assembly may still be hot after the Zymark Twister Microplate Handler loads it into the Plate Handler s output stack Please wait at least 30 seconds before manually handling the Snap On Compression Pad plate assembly 6 Choose from the following If performing Absolute or Relative Quantification or Dissociation Curve Analysis Run the plate as explained in Running the Plate on page 4 25 Allelic Discrimination 4 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Preparing Op
436. tem and SDS Enterprise Database User Guide Bar Code Readers Bar Code Readers Description The Applied Biosystems 7900HT Fast Real Time PCR System includes two bar code readers for data entry and plate recognition e A hand held bar code reader for scanning plates manually e A fixed position bar code reader for automatically scanning plates as they are loaded into the instrument available only with the Automation Accessory Both bar code readers use a 670 nm Class II laser to scan plates and are capable of reading Code 128 alphanumeric which supports 128 ASCII character bar codes Locations of the Bar Code Readers Fixed Position Bar Code Reader Hand Held Bar Code Reader To computer Splitter keyboard port aot To keyboard GR2015 Figure 1 5 Locations of the Bar Code Readers of the 7900HT Instrument Using the ANIA LASER HAZARD Exposure to direct or reflected laser light Bar Code can burn the retina and leave permanent blind spots Never look into the laser beam Readers Remove jewelry and anything else that can reflect the beam into your eyes Protect others from exposure to the beam For more information on the bar code readers see Using the Hand Held Bar Code Reader 0 000 000 ce
437. ter used to operate the 7900HT instrument vary with the version of the SDS software To determine the system requirements for your instrument check the release notes accompanying your version of the SDS software The release notes for the SDS software are in the following location D AppliedBiosystems SDS2 2 1 ReleaseNotes txt Note You can use the Microsoft Notepad Wordpad or Word software to read the release notes text file During installation of the 7900HT instrument the Applied Biosystems service engineer partitions the computer hard drive to create the logical drives shown in the table below Primary Hard Drive Drive ao Contains C 2 Operating system files D gt 25 e SDS Software e Automation Controller Software e Zymark Twister Software e LAVA Softwaret e Additional Third Party Software e SDS 7900HT Documents e Optional SDS Enterprise Database Client Software Applied Biosystems recommends that you do not install programs to the C drive The computer will boot faster if the C drive contains only the operating system tSoftware required for the operation of the bar code reader Note The partitions shown above represent the basic drive architecture required by the SDS software The actual number and size of the partitions and drives available on your computer vary depending on the model of computer accompanying your instrument 1 6 Applied Biosystems 7900HT Fast Real Time PCR Sys
438. ternative to the option 4 36 using the Template Batch utility above the software includes a IMPORTANT If using the Template Batch utility that can simultaneously create multiple plate documents from the plate document template SDS Enterprise Database you must choose this option Creating a SDS 1 Inthe SDS software click or select File gt New Plate Document from a Template 2 Configure the New Document dialog box e Assay drop down list Select the same assay as the plate document template e Container drop down list Select the same plate type as the plate document template e Template drop down list Select the plate document template sdt created on page 3 22 If the plate document template does not appear inside the Template drop down list select the file as explained below a Click Browse l b In the Look in field of the Open dialog box navigate to and select the plate document template sdt created on page 3 22 c Click _ open The Template drop down list displays the plate document template Note The Template drop down list displays all plate document templates contained in the Templates subdirectory of the SDS program directory e Barcode field Optional Do one of the following Enter the bar code number for the plate or Scan the plate bar code using the hand held bar code scanner 3 Click __ to create a new plate document from the plat
439. the aluminum foil backing is damaged creased bent or folded After removing the Low Density Array from its packaging dust or other particulates settle on the reaction wells optical side of the Low Density Array After removing the Low Density Array from its packaging water condenses on the reaction wells optical side of the Low Density Array After pipetting too little PCR reaction mixture has gone into the fill reservoir After pipetting some of the PCR reaction mixture leaks out of the vent port in the fill reservoir The PCR reaction mixture leaks during PCR cycling After a mean assay result and standard deviation of the replicates are calculated a specific replicate appears to be an outlier SDS Enterprise Database Archive or restore operation was terminated 8 20 prematurely Cannot Connect to the Database 8 4 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Low Precision or Irreproducibility Low Precision or Irreproducibility Overview Improper Threshold Setting There are many reasons why an assay run with the Applied Biosystems 7900HT Fast Real Time PCR System can have less than optimal precision Factors that can affect precision are described in detail below Improper Threshold SOU 065 5 boon deed coe de nebawhepesewedtaeedads 8 5 imprecise FOUN cece oc beet eraa ara be GRR ewe be ae tee eS eee 8 6 No on Optimized Chemisty coo sc
440. the plate contacts the sides of the stack re adjust the rotary position of the Plate Handler arm until the plate moves freely in the stack Note Contact between the plate and the stack or all stacks may be unavoidable However try to minimize the contact as much as possible 10 Using the Vertical Positioning commands raise and lower the Plate Handler arm several times to check the alignment 11 Lower the Plate Handler arm to the bottom of the plate stack click Ta Rotary offset and click es The software records the rotary position for the Zymark position 4 input stack 1 12 Click Open Gripper The gripper releases the plate Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 43 Chapter 7 Maintaining the Instrument Aligning the Plate Handler to the Instrument The next step is to align the Plate Handler arm to the instrument tray Zymark position 2 This alignment will ensure a smooth exchange between the Plate Handler arm and the instrument tray during operation of the instrument Note The following procedure requires you to position the Plate Handler relative to the 7900HT instrument Before moving the Plate Handler loosen the three black thumbscrews on the platform connecting the 7900HT instrument and the Plate Handler 1 Ifnot already present place an empty plate into input stack 1 Zymark position 4 and pick it up with the Plate Handler arm a In the Zym
441. the plate in terms of markers The well marker information consists of two sections the Well Marker List Header and the Well Marker Definition List 12 Well Marker List Header Allelic Discrimination Only IMPORTANT Well Marker List Header information is required only in setup table files created for allelic discrimination runs This section does not appear in setup table files created for absolute or relative quantification runs Description This line contains the column headings for the well marker information section of the setup table file that make the file easier to edit using a program such as Microsoft Excel Format Well lt tab gt Sample Name lt tab gt Detector lt tab gt Task lt tab gt Quantity lt tab gt Detector lt tab gt Task lt tab gt Quantity lt cr gt Example Example Code A 11 Well Sample Name Marker Task Marker Task 13 Well Marker Definition List Allelic Discrimination Only IMPORTANT Well Marker Definition List information is required only in setup table files created for allelic discrimination runs This section does not appear in setup table files created for absolute or relative quantification runs Description This section defines the contents of the plate wells The setup table file must contain a definition for each well used on the plate Each well definition list consists of one string of characters terminated by a lt cr gt The definition consists of three main divisions
442. thernet 100Base T network environment The database can function as part of an isolated star network via the SMC router provided with the database Figure 1 14 or as a node on a larger bus network Figure 1 15 The server computer uses a static IP address to simultaneously serve up to 5 clients configured for either DHCP or static TCP IP operation 1 22 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide About the SDS Enterprise Database Feature LAN Architecture Example In a basic configuration the SDS Enterprise Database can serve up to four DHCP or TCP IP clients configured in a star topography In this configuration the server is linked to the clients via the router provided with the database which has been configured with a static IP address Note The router features a WAN connection for connecting to an external bus Data Collection Analysis e Run Data e Analysis Sessions e Analysis Sessions e Run Data SDS Enterprise Database e Studies e New Plate Documents e Template Documents e Existing Plate Documents e Template Documents Applied Biosystems Database Server Desktop Computers Opertaing System 7900HT Fast Real Time Windows 2000 PCR System instruments Windows 2000 Network Protocol TCP IP Port 1521 TCP IP m ais SDS Enterprise Client Software Database Software SDS Client Software or Automation Controller Software Router Opertaing S
443. tical Plates for Use a Load the plate onto a designated thermal cycler and perform the PCR b Briefly centrifuge the plate to draw the reactions to the bottom of the wells and to eliminate any air bubbles that may have formed during thermal cycling c Run the plate as explained in Running the Plate on page 4 25 Cc 2 v V pue Q O O U _ La O C ia N pad for single plate runs Optical Caps flat caps only Snap On Compression Pad for automated runs Optical adhesive cover Figure 4 3 96 Well Optical Reaction Plate Assembly 2 on xe cq O Q pur Q O cover 384 well y ovo 100 000 000 000 000 00 030 030 020 080 080 080 4 384 Well Optical Reaction Plate Assembly Figure 4 4 9 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Chapter 4 Operating the Instrument Preparing TaqMan Low Density Arrays for Use About the Low Density Arrays How It Works Low Density Array Components The TaqMan Low Density Arrays are research tools for profiling gene expression using the Comparative Cy Method of relative quantification The Low Density Array evaluates from one to eight cDNA samples or controls generated from human total RNA in a two step RT PCR experiment The Low Density Array functions as an array of reaction vessels for the PCR sequence detection step Typically the wells of the
444. tinually develops the SDS software to provide increased functionality and reliability of the Applied Biosystems 7900HT Fast Real Time PCR System As updates become available Applied Biosystems sends notifications of the upgrades to all Applied Biosystems 7900HT Fast Real Time PCR System customers If an upgrade is user installable it can be found on the Applied Biosystems Company Web site see How to Obtain Services and Support on page xii Note Applied Biosystems service engineers perform regular software updates during planned maintenance visits On rare occasions when a piece of the SDS software becomes corrupt 1t may be necessary to re install the software In the event that the software must be re installed observe the following guidelines to re install or upgrade the software e Unless instructed to do otherwise remove the SDS software using the uninstall utility Do not delete the program subdirectory from the Program Files directory e Install the SDS software under a user login that has administrator privileges on the computer e Unless instructed to do otherwise re install the SDS software to the same directory as the previous installation e Review all documentation accompanying the new software such as installation notes or user bulletin The updated version of the software may contain new features that require special consideration Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database Use
445. tion of the 7900HT Instrument Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 17 Chapter 1 Product Overview Automated Operation In this mode the Applied Biosystems 7900HT Fast Real Time PCR System uses an Automation Accessory Zymark Twister Microplate Handler and fixed position bar code reader to provide high throughput operation suitable for small to medium scale studies With this configuration Figure 1 12 a technician can run batches of plates unattended using the Automation Controller Software As in Stand Alone operation the computer stores all data for operation of the instrument and provides the software for analyzing the run data Computer SDS Software Plate Documents Automation Controller Created Software Hard drive s Plate document files added to and run from Applied Biosystems 7900HT the plate queue Fast Real Time PCR System Instrument Firmware Raw Data Saved to the Plate Documents Thermal Cycling and Sequence Detection Plate Document Dat llect ata Collection Opened and Analyzed Figure 1 12 Automated Operation of the of the 7900HT Instrument Database Operation For this mode the Applied Biosystems 7900HT Fast Real Time PCR System uses the SDS Enterprise Database and the Automation Accessory to provide maximum high throughput potent
446. tion plate into input stack 1 of the Plate Handler Repeat steps 6 through 7 until the Plate Handler arm successfully retrieves the plate Grasp the plate with one hand and from the Zymark Twister Software Click Open Gripper to release the plate Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 39 Chapter 7 Maintaining the Instrument 10 Exit the Zymark Twister Software a Click Main Menu b Click Quit Application to close the software A defect in the Zymark Twister Software can cause portions of the program to persist in memory even after the software has been closed Because the Zymark Twister Software conflicts with the SDS software the residual elements of the software must be closed inside the Windows Task Manager before continuing 1 Confirm that the stack has closed by viewing the Task Manager a Press the Crtl Alt Del keys in unison b In the Windows Security dialog box click Task Manager c In the Task Manager dialog box confirm that the software has closed by looking for the Zymark Twister Software entry in the Task list If the software is still running click the software entry and click Close to exit the remaining software d Select File gt Exit 7 40 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Aligning the Plate Handler Aligning the Plate Handler When to Perform Preparing the Instrument f
447. tions 7 Number of Markers Allelic Discrimination Only see page A 8 8 Markers List Header Allelic Discrimination Only see page A 8 9 Markers List Allelic Discrimination Only see page A 9 Well Detector Information 10 Well Detector List Header see page A 10 11 Well Detector Definition List see page A 10 Well Marker Information 12 Well Marker List Header Allelic Discrimination Only see page A 12 13 Well Marker Definition List Allelic Discrimination Only see page A 12 The following conventions are used in the rest of this section e courier Text appearing in bolded courier font must be applied to a setup table file exactly as appears in this document e italic Text appearing in italic courier font must be substituted with custom values when applied to a setup table file e required text Text appearing between brackets is required information in setup table files All information inside the brackets must be present in the setup table file for the SDS software to import it e optional text Text appearing between braces is optional in setup table files e lt tab gt The tab character the equivalent of pressing the Tab key e lt cr gt The carriage return character the equivalent of pressing the Enter key IMPORTANT To guarantee a successful importation of the setup table file into a plate document the file must contain all the elements described in the following section and
448. to correctly pipette the PCR reaction mixture 100 uL into the fill reservoir 1 Carefully follow the procedures provided in Loading the TaqMan Low Density Arrays on page 4 14 2 After centrifuging be sure to note the fluid levels in the fill reservoir as described in step 6 on page 4 18 The PCR reaction mixture leaks during PCR cycling The aluminum foil backing may have been damaged during the sealing step Discard the Low Density Array Note See About the Sealer on page 4 14 for proper sealing instructions After a mean assay result and standard deviation of the replicates are calculated a specific replicate appears to be an outlier N A Review the multicomponent analysis display for that replicate Delete the outlier and reanalyze Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 8 19 Chapter 8 Troubleshooting SDS Enterprise Database SDS Enterprise Table 8 7 Troubleshooting the SDS Enterprise Database Database Troubleshooting Observation Possible Cause Recommended Action Table Cannot Connect to the N A To test or reset the database Database connection 1 Start the SDS software 2 Log into the database as a user with Administrative privileges 3 Test and reconfigure the database connection as described in Reconfiguring the Database Connection on page A 23 Error message Rare error
449. tomated Operation As with the SDS Software the Automation Controller Software requires the creation of a plate document for each plate it runs The Automation Controller Software can run batches of plates using plate documents saved to the hard drive of the computer attached to the instrument or stored on an SDS Enterprise Database Follow the appropriate procedure for your system Operating the Software with an SDS Enterprise Database 4 32 Operating the Software without an SDS Enterprise Database 4 34 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 31 Chapter 4 Operating the Instrument Operating the Software with an SDS Enterprise Database Overview Running the Automation Controller Software with the SDS Enterprise Database is the least complicated option for operating the Applied Biosystems 7900HT Fast Real Time PCR System Plate documents saved to the SDS Enterprise Database are automatically queued set up for operation for use on the Automation Controller Software Following plate document creation plates can be prepared and loaded immediately onto the Zymark Twister Microplate Handler for use Workflow 1 Start the Automation Controller Software and log in to the database see page 4 32 2 If desired set the study associations for the relative quantification and allelic discrimination runs See page 4 33 3 Load prepared optical
450. ton the Cards START button _ Fr The centrifuge starts then automatically stops after 1 min per the programmed sequence 2 Repeat step 1 so that the Low Density Arrays are centrifuged for a total of two consecutive 1 min spins to ensure complete distribution of the sample specific PCR reaction mix 3 Open the centrifuge cover by pressing the OPEN button Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 4 17 Chapter 4 Operating the Instrument 4 When the cover has fully opened remove the buckets from the centrifuge then remove the card holders from the buckets 5 Remove all Low Density Arrays from the buckets by gently lifting them by their carrier sides 6 Examine the Low Density Arrays to determine whether filling is complete The amount of cDNA sample or control remaining in the fill reservoirs should be uniform and consistent from reservoir to reservoir e If there is excess cDNA sample or control remaining in a fill reservoir as shown below filling is incomplete Proceed to the next step A e Ifa fill reservoir is completely drained as shown below it is possible that some wells were not filled properly The Low Density Array should not be processed further Use another Low Density Array Alternatively you can process the Low Density Array further however you should void the results for the affected fill reservoir sample
451. top baseline cycle If you are inspecting an AutoCalled detector verify that the baseline is called correctly by the software and proceed to step 8 If the baseline is set incorrectly open the Analysis Settings dialog box configure the detector for manual calling then click __ ok After the software adjusts the data manually set the baseline and threshold for the detector as described in this procedure If the amplification plot looks like Then 2 000 EH The amplification curve begins after the default maximum baseline cycle 15 Adjustment of the baseline is not required 1 000 E 1 3 254 600 7 o 4 4 6 8 0 12 14 16 18 20 2F 24 26 28 30 32 34 36 38 40 2 000 EH The maximum baseline is set too high Decrease the Stop baseline value by dragging the right range marker amp to an earlier cycle 1 000 E 1 3 259 dtd o 2 4 6 8 10 12 14 16 fe 20 22 24 2 28 30 32 34 36 38 4D 2 000 EH The maximum baseline is set too low Increase the Stop baseline value by dragging the right range marker to a later cycle 1 000 E Ar 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 8 Double click the Amplification plot or click Bj Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 47 Chapter6 Analyzing Real Time Data 9 In the Display Settings dialog box a In the Select Pane View field select Amplification Plot
452. tors Description This line defines the total number of detectors on the plate Format Number of Detectors lt tab gt number of detectors lt cr gt Example xxx Number of Detectors 5 5 Detectors List Header Description This line contains the column headings for the Detector Definitions section of the setup table file that make the file easier to edit using a program such as Microsoft Excel Format Detector lt tab gt Reporter lt tab gt Quencher lt tab gt Description lt tab gt Comments lt cr gt Example Example Code A 1 Detectors List Header Detector Reporter Quencher Description Comments A 6 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 Detectors List Setup Table Files Description The detector list consists of one or more lines displaying the information for each detector used on the plate document e The Detectors List section must contain one line or definition for each detector present on the plate e The number of lines in the Detectors List section must be equal to the number defined in the Number of Detectors section see number 4 above e Leave blank the Quencher Dye entry for detectors created for the SYBR Green I Dye or probes labeled with a non fluorescent quencher Format fora detector name lt tab gt reporter dye lt tab quencher dye lt tab gt single detector Examples description lt tab gt comments lt cr g
453. tral Calibration Kit PN 43823977 4331405 7900HT System Standard 96 Well Block Upgrade Kit 1 kit Includes a Standard 96 Well Block a 96 well plate adapter and an ABI PRiSM 7900HT Sequence Detection Systems 96 Well Spectral Calibration Kit PN 43828639 4351402 7900HT System Fast 96 Well Block Upgrade Kit 1 kit Includes a Fast 96 Well Block a Fast 96 Well Plate Adapter a 7900HT System Fast 96 Well Spectral Calibration Kit PN 4351653 a TaqMan RNase P Fast 96 Well Instrument Verification Plate PN 4351979 and a TaqMan Fast Reagents Starter Kit PN 43852407 4329012 7900HT System TaqMan Low Density Array Upgrade 1 kit C 2 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Consumables and Disposables Consumables and Disposables The Applied Biosystems 7900HT Fast Real Time PCR System can run e ABI PRISM 384 Well Optical Reaction Plates sealed with optical adhesive Covers e ABI PRISM 96 Well Optical Reaction Plates sealed with optical adhesive covers or ABI PRISM Optical Caps flat cap strips only e Optical 96 Well Fast Thermal Cycling Plates sealed with optical adhesive Covers e TaqMan Low Density Arrays The optical plates recommended above are designed specifically for fluorescence based PCR chemistries and are frosted to minimize external fluorescent contamination Before running prepared optical plates on the 7900HT inst
454. trument absolute and relative are described in detail below Absolute The objective of an absolute quantification experiment is to accurately determine the Quantification absolute quantity ofa single nucleic acid target sequence within an unknown sample The results of an absolute quantification experiment are reported in the same units of measure as the standard used to make them Relative The objective of relative quantification is to determine the quantity of a single Quantification nucleic acid target sequence within an unknown sample relative to the same sequence within a calibrator sample Relative quantification of target gene expression 1s calculated from Applied Biosystems 7900HT Fast Real Time PCR System data using one of the following methods About the Standard Curve Method The Standard Curve Method constructs a standard curve similar to that used in absolute quantification experiments However because the goal of relative quantification is to establish a fractional relationship data produced by relative quantification standards is used differently For all experimental samples target quantity is determined from the standard curve and divided by the target quantity ofa calibrator sample Because the unit from the standard curve drops out only the relative dilutions of the standard are necessary to determine relative quantities About the Comparative C Method The SDS software calculates relative levels of gene expression using
455. tton to have the software calculate a baseline for the selected detector s Manual Baseline option button and enter Start and End cycle values to apply to the selected detector s Manual Baseline option button and leave the Start and End cycles fields empty You will need to set the values manually as explained on page 6 46 4 If necessary repeat steps 2 through 3 for any additional detectors IMPORTANT The threshold and baseline values set in step 3 on page 6 11 are detector specific and must be set for each individual detector used on in the plate document 5 Click to accept the analysis settings and exit 6 Analyze the data as explained below Analyzing the After you have configured the analysis settings you can analyze the run data During Run the analysis the software mathematically transforms the raw data to establish a comparative relationship between the spectral changes in the passive reference dye and those of the reporter dye Based on that comparison the software calculates a cycle threshold Cy for each reaction standard and unknown The software then generates a standard curve for the run by plotting the standard samples on a graph of Cy versus initial copy number Note See Appendix D Theory of Operation for a detailed description of the SDS software mathematical transformation of real time run data 1 Click or select Analysis gt Analyze The SDS software analyzes the run data and disp
456. tware allows you to limit the detector and plate data displayed in the Gene Expression plot through the Plates and Targets tables of the Plate List Panel The software displays data from the targets and plates in the lists whose check boxes are selected About the Gene The SDS software displays the graphic equivalent of the results of the relative Expression Plot quantification AAC calculations in the Gene Expression plot The plot is in the upper division of the multiple plate document Figure 6 12 illustrates the important components of the plot Error bar see below aS Arrow see page 6 31 Gene Expression Plot Legend Plot Detectors Figure 6 12 Components of the Gene Expression Plot e X Axis Lists all the targets involved in the analysis Within each target group the software displays the relative quantity of the target in each sample e Y Axis The results of the relative quantification calculations are grouped by target sequence and the relative quantities are graphed on a logarithmic scale The quantities are shown relative to the expression level in the calibrator sample where each increment corresponds to a 10 fold difference in gene expression About the The SDS software displays the results of the relative quantification calculations on a Expression logarithmic scale where each increment corresponds to a 10 fold difference in gene Levels expression The software displays the expression level for eac
457. tween the statements in the Add Files dialog box Logical IF Statements By default the first statement in the table is always an IF statement If a plate document meets the criteria defined by the statement it 1s displayed within the Add Files dialog box Logical AND Statements A logical AND statement evaluates the data set for two expressions the expression defined by the AND statement and the expression defined by the statement in the previous table row When an AND statement is used in a filter table the software displays a plate document if it evaluates true for both expressions Logical OR Statements Similar to the logical AND statement a logical OR statement also evaluates the data set for two expressions both the previous and current table rows When an OR statement is used in a Filter table the software displays a plate document if it evaluates true for either expression or both Multiple AND and OR Statements If dealing with a very large data set you may want to further refine the number of plate documents retrieved in a search by creating multiple filter statements When creating multiple statements remember to create and apply them one at a time to ensure that they function as expected Also remember that the software applies each filter in the order listed in the Add Files dialog box Column Each statement evaluates plate documents based on the values of specific plate Categories document attributes Condit
458. ule e Initiates and controls the sequence detection run e Acquires data during the run Zymark Twister Software Used to calibrate the Zymark Twister Microplate Handler LAVA Software Used to align the fixed position bar code reader SDS Enterprise Database Links the SDS software to an SDS Enterprise Database Client Software on the network Note See page 1 21 for more information on the software components of the SDS Enterprise Database 1 14 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide SDS Software Related Files and Formats SDS Software Related Files and Formats Files Used and IMPORTANT Never move or delete program files or directories for SDS related Created by the software unless specifically directed to do so by an Applied Biosystems SDS Software representative or documentation The SDS software uses many different files and directories Table 1 5 Some of these files store run data and calibration data others are required to run the 7900HT instrument Table 1 5 Common SDS Files File Type Ext Icon Description Comments Plate Document Files SDS 7900HT sds ma Serves as a virtual representation of a single Document LR consumable used to contain samples and reagents during a sequence detection run e Contains all sample and assay data names locations reporter dye contained by the consumable e Contains instructions for running the instrument thermal cycling and
459. up 1 23 database WAN setup 1 24 Archiver 1 26 archiving files 7 54 arrows sample bars 6 31 assay development guidelines absolute quantification B 2 to B 4 B 6 allelic discrimination B 2 to B 5 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Index 1 relative quantification B 6 automatic outlier removal 6 19 Automation Accessory components 7 36 Plate Handler See Plate Handler See fixed position bar code reader Automation Controller Software about 4 39 adding plates to the plate queue 4 40 configuring for operation 4 42 ejecting a plate 4 44 launching 4 39 monitoring instrument progress 4 44 removing plates from the plate queue 4 40 starting the plate queue 4 43 stopping the plate queue 4 44 B background run 7 16 to 7 19 about 7 16 constructing a background plate 7 17 creating a plate document 7 17 extracting 7 19 preparing a background plate 7 17 troubleshooting 8 9 when to perform 7 16 bar code information entering into a plate document 2 27 3 25 bar code readers fixed position See fixed position bar code reader hand held See hand held bar code reader bars in the Gene Expression Profile symbols 6 31 baseline about D 6 configuring value for automatic analysis 6 11 Block readout from the Real Time tab 4 27 Bucket Type setting on centrifuge 4 13 C calibrating the 7900HT instrument adjusting the plate sensor switch 7 37 aligning the fixed position bar
460. ure 6 11 Gene Expression Settings for Study Settings Calibrator Sample Displays the name of the sample that the software uses as the calibrator for the study Endogenous Control Detector Specifies the name of the detector that the software will use as the endogenous control for the study Control Type The Multiplexed Non Multiplexed option buttons specify how the software will use the data from the wells labeled with the endogenous control data RQ Min Max Confidence Specifies the confidence value that the software will use to calculate the standard error of the mean expression level RQMax and RQMin values for the samples in the study Export Individual AC s Instructs the software to export the AC values of individual samples in the analysis instead of the average AC values for the replicate group Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Analyzing the Study Specifying the Analysis Settings Specifying the 1 Click or select Analysis gt Analysis Settings Settings for the 2 In the Detector drop down list select Analysis e detector name To set the Analysis Settings for a specific detector e All Detectors To set the Analysis Settings for a all detectors simultaneously 3 Inthe Analysis Settings dialog box configure the method automatic or manual that the software will use to determine the baseline and threshold values for the select
461. ures described below see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide The Reason s for Change dialog box appears only if the SDS Enterprise Database is configured for auditing The auditing system monitors the creation modification and deletion of the SDS data contained by the database When the Reasons for Change dialog box appears do one of the following e Select a description for the change from the drop down list then click ox e Enter a description of the reason for the change in the field then click Reasons for change drop down list Resson For Changa Atirbuts AGPiata Cid Vetus Hew Value Default descriptions Calculation Error Meed to Change Threshold Example of a custom description for a change Figure 4 1 Reasons for Change Dialog Box Options Erie ihe Reaconor Change Custom description of the reason for the change The Electronic Signature Verification dialog box appears only if the SDS Enterprise Database is configured to require electronic signatures The electronic signature system restricts and tracks user access of the SDS data contained by the database When the Electronic Signature Verification dialog box appears 1 In the User ID and Password fields enter your user name and password then click 2 After the software validates your user name and password it displays a message stat
462. ve quantification plates in a study you must first add the plates to a Relative Quantification Multiple Plate Document Plate documents added to a study must Be of the same consumable format for example you cannot compare samples run on 384 well plates to those run on 96 well plates Have been run using the same method identical thermal cycler conditions sample volume and data collection options Contain sample names for all In Use wells If a plate document does not contain samples you can apply sample names to it using the SDS software IMPORTANT Relative Quantification Multiple Plate Documents exclude from the analysis all wells of plates that do not have a sample name applied to them IMPORTANT Relative Quantification Multiple Plate Documents cannot compare data from runs performed using different plate formats for example samples run in a 96 well plate cannot be compared to those run in a 384 well plate To add plates to the Relative Quantification Multiple Plate Document l 2 In the multiple plate document click _ AddPiatets In the Add dialog box do one of the following If you know where your plate documents are located a Select the File tab b Navigate to the directory containing the relative quantification plate document files of interest c Press and hold the Ctrl key select the files of interest then click Add Plates To Be Added To Study C pplied BiosystemstSDS Documents
463. w exponentially This signals the third stage of R change which is roughly linear and finally reaches a plateau at about 10 M Martens and Naes 1989 The progressive cleavage of TaqMan fluorescent probes during the PCR makes possible the correlation between initial template concentration and the rise in fluorescence As the concentration of amplified product increases in a sample so does the R value During the exponential growth stage the geometric phase the relationship of amplified PCR product to initial template can be shown in the following equation N N 1 E C where N is the concentration of amplified product at any cycle N is the initial concentration of target template E is the efficiency of the system and c is the cycle number The Applied Biosystems 7900HT Fast Real Time PCR System creates quantifiable relationships between test samples based on the number of cycles elapsed before achieving detectable levels of fluorescence Test samples containing a greater initial template number cross the detection threshold at a lower cycle than samples containing lower initial template The SDS software uses a Threshold setting to define the level of detectable fluorescence The threshold cycle C for a given amplification curve occurs at the point that the fluorescent signal grows beyond the value of the threshold setting The C represents a detection threshold for the 7900HT instrument and is dependent on two factors e
464. when Reattempt the archive or archiving or restoring a restoration task large data set java sql SQLException Io exception End of TNS data channel 8 20 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Software Reference In This Appendix Importing Plate Document Setup Table Files 00 000 cee eee A 2 el SO Eo ee aah ea os E ewe ee ee aes v4 OSs ede een eee es A 4 POG WO 4 5 64 oie ob oe GA 8 44 4s oh 0 ee ees A 16 Exporting Plate Document Data oy 50s ohn ee ieee anes een esas Enip e A 16 Operating the SDS Software from a Command Line A 18 Usame ie Sea DOl non ses Hee dee eeenee chee dees 4004604550 0009405 A 20 Connecting SDS Software to the Database 000 0c ee eee A 22 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide A 1 Appendix A Software Reference Importing Plate Document Setup Table Files About the Import Function Creating and Importing Setup Table Data into a Plate Document A 2 The SDS software features the ability to import setup table information detector detector task marker and sample name layouts into a plate document from a tab delimited text file The import feature is designed to be a timesaving device that facilitates the exchange of setup information between other programs and the SDS software Instead of setting up plate documents individually a thi
465. wing To set the analysis settings for e A single marker select a marker of interest from the Marker drop down list e All markers select All markers from the Marker drop down list 3 Inthe Analysis Settings dialog box a Select the Auto caller enabled check box to activate the AutoCalling system for the associated marker b If you expect the analyzed data to consist of only two clusters select the 2 Cluster Calling check box c Enter a percentage value in the Quality field to apply as the quality interval for AutoCalling samples 4 Repeat steps 2 through 3 for the remaining markers on the plate document 5 When finished click x During the analysis the software employs several algorithms to generate from the raw data a more direct measure of the relationship between the spectral changes in the samples To analyze the data 1 Click or select Analysis gt Analyze The SDS software analyzes the run data and displays the results in the Results tab 5 12 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Calling and Scrutinizing Allelic Discrimination Data Calling and Scrutinizing Allelic Discrimination Data About the Allelic The SDS software graphs the results of allelic discrimination runs on a scatterplot Discrimination contrasting reporter dye fluorescence After signal normalization and View multicomponent analysis the software graphs the normalized data from each well
466. wing analysis of the run data See Setting the Baseline and Threshold Values on page 6 46 for more information Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 8 5 Chapter 8 Troubleshooting Imprecise Pipetting Non Optimized Chemistry Incomplete Mixing Air Bubbles Splashing PCR Reagents The calculated quantities of target nucleic acid are directly affected by how precisely the template volumes are added to the reaction mixes Other individually added reagents are also affected by pipetting precision such as variable magnesium affects amplification efficiency Using Master Mixes For this reason Applied Biosystems highly recommends using a master mix All common components to a set of reactions should be mixed together and then dispensed to the wells of the plate Sub master mixes can be used to further improve the precision of identical replicates For example instead of pipetting 5 uL of the same template into four replicate wells pipette 20 uL of the template into a sub master mix then divide the sub master mix into four equal parts for amplification When making each master mix add 5 10 additional volume to compensate for pipetting losses Using Pipettors Pipetting precision is also improved by e Calibrating and servicing the pipettors regularly e Pipetting larger volumes e Reducing the number of pipetting steps whenever possible e Increasing the consistency
467. wister Microplate Handler see Powering On the 7900HT Instrument 0 0 0 0 cee ees 2 4 Starting and Configuring the Automation Controller Software for Operation 4 39 Maintaining the Plate Handler c 440 c0c40ceeddea aeed eee beeeaadesaaas 7 35 1 8 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Compatible Consumables Compatible Consumables Consumables for Use with the 7900HT Instrument Compression Pads for the ABI PRISM 96 Well Optical Reaction Plates Consumable Requirements The interchangeable sample block modules of the 7900HT instrument allow it to support a variety of consumable formats listed in Table 1 1 Table 1 1 Compatible Consumables Consumable Compatible Seals ABI PRISM 384 Well Optical e ABI PRISM Optical Adhesive Covers Reaction Plates quantity 100 e Optical Adhesive Covers quantity 25 ABI PRiSm 96 Well Optical e Compression Pads Reaction Plates Standard pad for manual operation ABI PRISM Snap On Compression Pad reusable e 96 Well Plate Seals ABI PRism Optical Adhesive Covers quantity 100 Optical Adhesive Covers quantity 25 ABI PRism Optical Caps flat caps only Optical 96 Well Fast Thermal e ABI PRISM Optical Adhesive Covers Cycling Plates quantity 100 e Optical Adhesive Covers quantity 25 TaqMan Low Density Arrays None self sealing To ensure
468. xamples in This The illustrations and screenshots that appear within this chapter were created from a Chapter plate run to determine the purity of a B actin amplification in unknown samples Each well of the plate contains SYBR Green 1 dye forward and reverse primers and genomic DNA known to contain complimentary binding sites Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 6 39 Chapter 6 Analyzing Real Time Data Analysis Checklist Workflow Overview 1 Analyze the run data see page 6 41 2 View the results of the dissociation curve analysis see page 6 42 Y 3 Determine melting temperature Tm values for the derivative peaks see page 6 44 4 Choose from the following post analysis options see page 6 44 Reanalyze the run data Adjust the display settings for the results table plate grid and plate document plots Print elements of the plate document Export the plate document results table or plots 6 40 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide Opening the Run Data Opening the Run Data User Access Requirement Open the Un analyzed Plate Document There is no access requirement All users can open dissociation curve data that has been saved to the SDS Enterprise Database Opening a Plate Document File l 2 3 4 Click or sele
469. xtraction in a dialog box If the software displays the e Calibration Update Complete dialog box then the run is successful Proceed to step 2 The raw spectra read from the Background plate conform to acceptable limits E Calibration Update Complete x i Background calibration information updated e Error dialog box then the run is unsuccessful Troubleshoot and decontaminate the sample block as explained in Background Runs on page 8 9 The software has stopped the extraction because one or more raw spectra exceed 2500 FSU x 3 The signal measured in some ofthe wells is too high and indicates block contamination This data is not usable for background extraction 2 Click J or select File gt Save The software saves the plate document 3 Select File gt Close The software closes the plate document Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 19 Chapter 7 Maintaining the Instrument Performing a Pure Dye Run When to Perform Purpose of Pure Dye Runs Components of the Pure Dye Spectra Applied Biosystems recommends performing spectral calibration e Every 6 months depending on instrument use e After changing sample block formats see page 7 6 IMPORTANT Always run a background plate before performing a Pure Dye calibration Pure dye data is generated from the results of a pure dye run in which the SDS software collects spectral d
470. y Components and Stack Positions 0 7 36 Adjusting the Sensitivity of the Plate Sensor Switch 0 7 37 Alanne ibe Piate on es eo oho Se ESS OOOO SS ER EER SS 7 41 Aligning the Fixed Position Bar Code Reader 0000 ce eee 7 49 Cleaning and Replacing Gripper Finger Pads 0 0 00 e ee eaee 7 52 Section 7 3 Maintaining the Computer and Software 00 7 53 General Computer Maintenance 0 ccc ees 7 54 Maintaining the SDS software 0 0 eens 7 55 Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 7 1 Chapter 7 Maintaining the Instrument Notes for Database Users Database Alerts and Notifications Reason s For Change Dialog Box Electronic Signature Verification Dialog Box If you are using an SDS Enterprise Database to store SDS plate documents sessions studies and data you may be required to perform additional tasks when performing the procedures described in this chapter This section describes the types of actions you may need to perform depending on how your administrator has configured the database For more information on any of the features described below see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real Time PCR System Administrators Guide The Reason s for Change dialog box appears only if the SDS Enterprise Database is configured for auditing The auditing system mo
471. ystem Windows 2000 Network Protocol TCP IP Client Software SDS Client Software RQ Manager Software or SNP Manager Software Figure 1 14 Basic LAN Architecture for the SDS Enterprise Database Applied Biosystems 7900HT Fast Real Time PCR System and SDS Enterprise Database User Guide 1 23 Chapter 1 Product Overview WAN Architecture Example The SDS Enterprise Database can serve up to 5 clients as a node on a bus network Again the server must be configured with a static IP address for identification on the network and is linked to the DHCP or TCP IP clients via the router provided with the database Data Collection Analysis e Run Data e Analysis Sessions e Analysis Sessions e Run Data SDS Enterprise e Existing Plate Documents Database e Studies e New Plate Documents e Template Documents e Template Documents Opertaing System Applied Biosystems Database Server Desktop Computers Opertaing System 7900HT Fast Real Time Windows 2000 PCR System instruments Windows 2000 Windows 2000 TCP IP Port 1521 SDS Enterprise Database Software Network Protocol TCP IP Network Protocol TCP IP Client Software SDS Client Software or Automation Controller Software Client Software SDS Client Software RQ Manager Software or SNP Manager Software Router Laboratory Office Figure 1 15 WAN Architecture for the SDS Enterprise Database 1 24 Applied Biosystems 7900HT Fast Real Time PCR System

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