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Absolute Quantitation Using Standard Curve Getting Started Guide

1. Plate Tab Displays the results data of each well including e The sample name and detector task and color for each well e A calculated value quantity default ARn or Ct Select Analysis gt Display to select the value to display Note For detectors without standards the Plate Tab displays Undet meaning undetermined Setup Instrument d A 1 1 1 1 1 1 1 1 1 2 1 2 1 2 1 2 M 4796 00 M 4 5se 00 M 4 826400 M 4 68e 00 M 4 94e 00 M 4 60e 00 M 4 62e 00 M 4 47e4 1 4 1 4 1 4 1 4 1 5 1 5 1 5 1 5 M 4 35e 00 M 4 s7e 00 M 4 20e 00 M 4 66e 00 M 4 79e 00 M 5 06e 00 M 4 75e 00 M 4 shes 1 7 1 7 1 7 1 7 1 8 1 8 1 8 1 8 M 4 74e 00 M 4 74e 00 M 5 08e 00 M 5 48e 00 M 5 24e 00 M 5 54e 00 M 5 23e 00 M 5 25e D NTC NTC NTC NTC 1 1 1 1 B 1 25e 00 H 1 25e 00 1 25e 00 H 1 25e4 s3 s3 s3 s3 S4 S4 S4 S4 B 5 00e 00 H 5 00e 00 B 5 00e 00 B 5 00e 00 B 1 o0e 00 B 1 o0e 00 B 1 00e 00 E 1 oes 2 1 2 1 2 1 2 1 2 2 2 2 2 2 2 2 M i o1e 00 M 9 90e 00 M 9 26e 00 M 9 70e 00 M 9 26e 00 M 1 02e 00 M 1 o4e 00 M 1 o2e RE R GR G 2 4 2 4 2 4 2 4 2 5 2 5 2 5 2 5 M i o3e 00 M 1 09e 00 M 9 49e 00 M 1 o0e 00 M 9 09e 00 M 9 61e 00 M 9 99e 00 M 1 o1es Spectra Tab Displays the fluorescence spectra of selected wells e The Cycles slider allows you to see the spectra for each cycle by dragging it with the pointer e The Cycle text box shows the current position of the slider
2. Notes Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 9 Chapter 2 Designing an AQ Experiment Overview Overview Typical AQ experiments are designed for traditional singleplex PCR where a primer pair plus a TaqMan probe or a primer pair plus a SYBR Green binding dye are present in a reaction The following sections describe design decisions required for AQ experiments Specifying the Components of an AQ Experiment For each AQ experiment specify e An unknown The nucleic acid sequence that you are quantitating e Standards This guide assumes that you have generated a set of standards for each target sequence that you are quantitating Appendix B on page 75 provides guidelines for generating standards e Replicate wells For absolute quantitation assays Applied Biosystems recommends the use of three or more replicate reactions per sample to ensure statistical significance For more information about these requirements refer to the Real Time PCR Systems Chemistry Guide PN 4348358 Example Experiment The example experiment determined the quantity of the RNase P gene in two populations on the 7500 Real Time PCR System Because a single gene was studied only one set of standards was required A Four replicates of each unknown and standard were performed to ensure statistical significance In experiments where multiple genes are being studied a set of
3. Source ABI PRISM 6100 Nucleic Acid PrepStation Applied Biosystems PN 6100 01 High Capacity cDNA Reverse Transcription Kit 1000 reactions High Capacity cDNA Reverse Transcription Kit 200 reactions High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor 1000 reactions High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor 200 reactions Applied Biosystems PN 4368813 Applied Biosystems PN 4368814 Applied Biosystems PN 4374967 Applied Biosystems PN 4374966 TaqMan Universal PCR Master Mix TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG Applied Biosystems PN 4304437 Applied Biosystems PN 4352042 TaqMan One Step RT PCR Master Mix Applied Biosystems PN 4309169 SYBR Green PCR Master Mix Applied Biosystems PN 4309155 Power SYBR Green PCR Master Mix Applied Biosystems PN 4367659 MicroAmp Optical 96 Well Reaction Plate Applied Biosystems PN 4306737 enn AEEA Applied Biosystems PN 4346906 MicroAmp Fast Optical 96 Well Reaction Plate with Barcode code 128 MicroAmp Optical Adhesive Film quantity 100 MicroAmp Optical Adhesive Film quantity 25 MicroAmp Adhesive Film Applicator Applied Biosystems PN 4311971 Applied Biosystems PN 4360954 Applied Biosystems PN 4333183 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast System
4. To export plate views or graphs as JPEG files 1 Select Tools gt Graph Export gt As JPEG alternately right click any graph or plate then select Export as JPEG Chapter 6 Analyzing AQ Data Exporting AQ Plate Data Export Settings Export only selected wells Apply Report Settings for Data Columns Cancel File View Tools Instrument Analysis Window Help OSa Run List Manager Ctrl Shift R Detector Manager Ctrl K Marker Manager Ctrl L and Cuna V Dissociation Y Dye Manager Ctrl Report Settings Standard Cum Graph Settings Document Information 24 Filter Configuration Graph Export gt E 4s JPEG Ctrl J To PowerPoint All to PowerPoint Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 11 Chapter 6 Analyzing AQ Data PEE Exporting AQ Plate Data The Export as JPEG dialog box opens Export As JPEG Select images you would like to save as JPEG files Select All Clear All Note In the Export as JPEG dialog box you can a CPE change default file names select image Elation Pet Mais Daya sd aegPocpa resolution and select which plate views or D Diesocsaion Mai Std Garve Disaster graphs to export and where the file s are saved e Refer to Online Help for more information about Type in fold
5. MV Show all standard points for selected detector s Detector RNase P iv 29 000 28 500 28 000 nknown Ct 27 500 27 000 Slope 3 577242 Intercept 41 033016 26 500 R2 0 997959 26 000 25 500 3 000 3 200 3 400 3 600 3 800 4 000 4 200 4 400 Log CO Displays the melting T curves associated with a dissociation assay The data are shown when using SYBR Green dye when either e Dissociation Protocol is selected in the Instrument tab e Dissociation is selected as the assay type Setup Y instrament YResuts Y Plate Amplification Plot Standard Curve Y Dissociation Dissociation Cure 0 16 Data Type 0 14 E Derivative r 0 12 0 10 Detector T oe SYBR Z 0 068 A 0 04 Tm 60 1 C 0 02 0 00 0 02 BO Temperature C Appendix C on page 77 and the Online Help provide information about dissociation curve analysis Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Report Chapter 6 Analyzing AQ Data eer l Analyzing and Viewing the AQ Data PRE Displays data for selected wells in tabular form The data columns associated with the report are determined by the assay being run For AQ assays the following data columns are available Well Sample Name Detector Task Ct StdDev Ct Quantity Mean Qty StdDev Qty Filtered Tm and three User Defined columns Refer to Online Hel
6. TACR2 FAM none 2003 0915 08 RARA FAM none 2003 0915 08 RNase P 3 FAM none 2003 09 30 13 RNase P 2 FAM none 2003 09 30 13 RNase P 1 FAM none 2003 09 30 13 RNase P FAM none 2003 09 30 13 KAN FAM none 2003 0945 08 IPC Netertnr Internal Positiw Wie fnane Annamaria lt ili gt Fie v Add To Plate Document New Duplicate Done Add to Plate Document Import Export Clear Clear All Properties Hew Detector Hame A 3 Description Zz Reporter Dye FAM Quencher Dye Color Notes Create Another Woaw Cancel Notes S Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 73 Appendix A Creating Detectors 5 8 9 In the Reporter Dye and Quencher Dye drop down lists select the appropriate dyes for the detector Note The dyes that appear on the Reporter and Quencher Dye lists are those that have been previously entered using the Dye Manager If the dye that you want to use does not appear in a list use the Dye Manager to add the dye and then return to this step in this procedure Refer to the Online Help for more information Note Select TAMRA as the dye quencher for TaqMan probes and None for TaqMan MGB probes Click the Color box select a color to represent the detector using the Color dialog box then click OK Optionally click the Notes field then enter any ad
7. Unknown 37 08 0481 596034 A411 RNaseP Unknown 37 78 0481 4907 72 5015 28 Notes 56 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 6 Analyzing AQ Data Adjusting the Baseline and Threshold ances f Setup Y netumeni Y Results Plate Amplification Plot Standard Curve Delta Rn vs Cycle 1 1 0e 001 Data Deka An ve Cycle l Detector RNase F slo lt 3 1 0e 000 Line Color well Color r Jee Exponential phase of the amplification curve Analysis Settings 1 0e 001 ee Manual Ct 5a m E hay 4 Threshold 0 728583 oOo ee ji L as er he 1 08 002 hef 5b f Auto Baseline Manual Baseline _ _4q T 1 0e 003 H Start cycle mas 4b End cycle i 5 AE 1 0e 004 To manually adjust the baseline and threshold 1 Select the Amplification Plot tab then select Delta Rn vs Cycle in the Data drop down list 2 Select the wells to display on the plot Otherwise the plot will be empty 3 In the Detector drop down list select a detector The SDS software displays the graph for the selected detector and wells Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 57 ae Chapter 6 Analyzing AQ Data ances Adjusting the Baseline and Thresh
8. lt Remove g New Detector lt Back Next gt Finish Cancel B sx 5K 5K 5K 5K 5K 5K 5K 5K 5K 5K 5K Oo D o o o o o o o o o D c K 5K 5K 5K 5K 5K 5K 5K 5K 5K 5K 5K oO o o D o o o o u o o o D nTc NTC NTC NTC s1 s1 s1 s1 2 2 2 2 H125e003 H125e003 H125e 003 1 25e 003 H 250e 003 H 250e 003 H 250e 003 H 2 50e 003 E s3 3 3 3 4 4 4 S4 55 55 55 s5 B 500e 003 H 500e 003 H 500e 003 H 500e 003 H 100e 004 H 1 00e 004 H 100e 004 H 1 00e 004 H 200e 004 H 200e 004 H 200e 004 H 2 00e 004 F 10K 10K 10K 10K 10K 10K 10K 10K 10K 10K 10K 10K o o o o o o o u o o o G 10K 10K 10K 10K 10K 10K 10K 10K 10K 10K 10K 10K oO oO o o o o o u u u u H 10K 10K 10K 10K 10K 10K 10K 10K 10K 10K 10K 10K oO o o o o o o o o o o o Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 85 Appendix E Example AQ Experiment 8 Start the AQ run f Setup Instrument Y Results 4 Instrument Control a Select the Instrument tab By default the standard PCR conditions for the PCR step Estimated Time Remaining hh mm of the two step RT PCR method are m displayed See the figure to the right The figure on page 86 shows the default PCR conditions for the 7500 Fast system Poua S Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 Stage 3 Reps Reps Reps b Select File gt Save enter
9. was added to each well The plate was kept on ice until it was loaded in the 7500 system Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 23 Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Creating an AQ Plate Document Creating an AQ Plate Document Overview An AQ plate document stores data collected from an AQ run for a single plate AQ plate documents also store other information about the run including sample names and detectors Run Setup For each AQ plate document that you create specify detectors standards and detector Requirements tasks e A detector is a virtual representation of a gene specific nucleic acid primer and probe combination used in assays You specify which detector to use for each target sequence Appendix A on page 73 explains how to create detectors e A standard is a known amount of a target sequence You must have a set of standards for each target sequence on the plate e A detector task specifies how the software uses the data collected from the well during analysis You apply one of three tasks to each detector in each well of a plate document Task Symbol Apply to detectors of Unknown m Wells that contain target sequences that you are quantitating Standard E Wells that contain samples of known quantities No Template M Negative control wells that contain PCR reagents but that Controls NTC lack templ
10. 57 Manual Baseline If you set the baseline and threshold values manually for any detector in the study you and Threshold must perform the procedure on page 57 for each of the detectors SEICA The following amplification plots show the effects of baseline and threshold settings 54 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 6 Analyzing AQ Data Adjusting the Baseline and Threshold FRE Baseline Set Correctly The amplification curve begins after the maximum baseline The threshold is set in the exponential phase of the amplification curve No adjustment necessary Delta An 5 7 9 11 13 15 17 19 71 23 25 327 39 J1 33 35 J J39 Cycle Number Baseline Set Too Low The amplification curve begins too far to the right of the maximum baseline Increase the End Cycle value Delta Rn Ta ise ie SW ee Sessa ee OP se Sa Sar eau Cycle Nurnber Baseline Set Too High The amplification curve begins before the maximum baseline Decrease the End Cycle value Delta Rn eee Se se eee ee oe Ses eee eee clea soos eae Cycle Number Notes Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 55 Chapter 6 Analyzing AQ Data ances Adjusting the Baseline and Threshold Threshold Set Correctly The threshold is set in
11. Dissociation W Standard Curve Mean Oty C Landscape 0 Landscape StdDev Oty Portrait Portrait Filtered Landscape f Landscape Click Chose Data Tro Choose Data Columns and Ordering Columns and Additional Data to Print in the Report Ordering for more C Show detector results in detector color report options W Document Comments lf Analysis Methods p p Show grapwhite rows W Thermal Profile W Detector Setup H of white rows 4 of Gray rows 4 OF Cancel Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 65 eee l Chapter 6 Analyzing AQ Data PEE Omitting Samples Omitting Samples Experimental error may cause some wells to be amplified 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 calculations these outlying data outliers can result in erroneous measurements To ensure precision carefully view replicate groups for outliers You can remove outliers manually using the Cy vs Well Position Amplification Plot or the Standard Curve plot Removing Outliers on the Amplification Plot 1 Select the Amplification Plot tab Setup Y mstument YResuts Y Amplification Plot Standard Curve Y Dissociation Y Report i Ct vs Well Position 2 Inthe Data drop down list select Ct vs Well re Positi
12. For example Type 0 then press Enter for each of the remaining fields e Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix e A right arrow bracket gt separates successive commands you select from a drop down or shortcut menu For example Select File gt Open User Attention The following user attention words appear in Applied Biosystems user documentation Words Fach 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 N or Ailo 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 Neen Indicates a potentially hazardous situation that if not avoided could result in death or serious injury Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems vii Preface How to Obtain More Information How to Obtain More Information Related For more information about using the 7300 7500 7500 Fast system refer to Documentation e Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Online Help Applied Biosystems
13. Plate Document 0 000 eee ee 38 Specifying Thermal Cycling Conditions and Starting the Run 43 TFOUDIGSHOOUNG 35 255 Sa aowkdeewees ites 3S ho aoe eh eee e 48 Analyzing AQ Data 51 Configuring Analysis Settings 20 0 0 cc eee eee 52 Adjusting the Baseline and Threshold 0 0000 cee ees 54 Analyzing and Viewing the AQ Data 0 0 cc eee 60 OMNO SAMD co eee ae ee ee ee ee ae ee a eee ere re eee 66 ExXpOning AQ Plate Dalavics cehtcwe hee brae tone aoe te hehe eeawe dee 70 Creating Detectors 73 Guidelines for Generating Standard Curves 75 Dissociation curve Analysis 1 Isothermal Assays 79 Example AQ Experiment 81 References 89 Index 91 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Preface How to Use This Guide Purpose of This This manual is written for principal investigators and laboratory staff who conduct Guide absolute quantitation assays using the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 7300 7500 7500 Fast system Assumptions This guide assumes that you have e Familiarity with Microsoft Windows XP operating system e Knowledge of general techniques for handling DNA and RNA samples and preparing them for PCR e A general understanding of hard drives and data storage file transfers and copying and pasting Text Conventions This guide uses the following conventions e Bold indicates user action
14. Refer to the Online Help for more information Users of the 7500 Fast System can use SYBR Green I dye with Standard or 9600 Emulation Run Modes IMPORTANT To enable Expert Mode continue to step 3 Otherwise skip to step 6 3 Click the Expert Mode checkbox 4 Click the Select View Filters button F Setup Yinstrument YResults 4 Instrument Control Temperature C smn Estimated Time Remaining hh mm Sample Heat Sink Cover Block Cycle Status Stage Rep Time mm ss Step State Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 ll Add Cycle Add Hold Add Step Add Dissociation Stage Help Settings Sample Volume pL 120 Run Mode Fast 7500 X Expert Mode Data Collection Stage 2 Step 2 60 0 0 30 v Settings Sample Volume pL 20 Run Mode Fast 7500 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 45 Chapter 5 Running an AQ Plate 7500 Fast System Specifying Thermal Cycling Conditions and Starting the Run 5 Select filters for data collection and click OK If Filter Selection Expert Mode the checkbox next to a filter is checked that filter Tite ng eae ee is used for data collection E Filter B IMPORTANT If no filter is selected no data will z kaji be collected Cancel Note The use of ROX dye is strongly recommended to normalize variation caused by pipetting erro
15. Setup Y instrument YResults Y Plate Y Spectra Y Component Y Amplification Plot Y Standard Curve Raw Data 1347146 00 1200000 00 1000000 00 800000 00 600000 00 Fluorescence 400000 00 200000 00 0 00 Cycle 40 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 61 Analyzing and Viewing the AQ Data PERCE Chapter 6 Analyzing AQ Data Component Tab Displays the complete spectral contribution of each dye in a selected well over the duration of the PCR run Only the first selected well is shown at one time Setup instrument Results Plate Spectra Y Component Amplification Plot Standard Curve Y Component 12000 Component s Mv Fam 10000 Mv Rox Mv TAMRA 8000 6000 ee H Sapapannnannannanoanynnn nast ALLL d ag ye 31 Fluorescence ui wet Sey ke WEL ea Fe Fi ee Fe Se Ste Sif ak Cycles 0 Note If you are using TaqMan products three components ROX dye reporter dye and TAMRA dye labeled quencher are displayed in the Component tab If you are using TaqgMan MGB products only two components ROX and reporter dyes are displayed Amplification Plot The three Amplification Plots allow you to view post run amplification of specific Tab samples The Amplification Plots display all samples in the selected wells Rn vs Cycle Linear View Displays normalized report
16. System Preparing the Reaction Plate 5 Verify that each reaction is positioned in the bottom of the well Correct Position Incorrect Positions m GR1303 U U The reaction is positioned The reaction lies on the An air bubble lies at the correctly in the bottom of side wall because the bottom of the well the well plate was not centrifuged because the plate was not centrifuged with sufficient force or for sufficient time IMPORTANT Ensure all reaction is positioned correctly in the bottom of the well before starting a run Failure to do so will impact the quality of data 6 Keep the reaction plate on ice until you are ready to load it into the 7500 Fast system 36 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 5 Running an AQ Plate 7500 Fast System Preparing the Reaction Plate Example Experiment The PCR master mixes were prepared according to the universal assay conditions 3 l uL 5 uL 37 Final FeacM COMPONENI VEREACHON Reactionst Reactions Concentration TaqMan Fast Universal PCR Master Mix 10 0 50 0 370 0 1X 2X TaqMan Gene Expression Assay Mix 20X e Forward PCR primer 18 uM 50 to 900 nM e Reverse PCR primer 18 uM Equal to 1 0 Equal to 5 0 Equal to 37 0 50 to 900 nM e TagMan probe 5 uM 50 to 250 nM CDNA sample or template for standards 10
17. TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG Most custom 5 nuclease quantitation assays designed with the Applied Biosystems Assay Design Guidelines will provide comparable performance when run using the default Fast thermal cycling conditions and the TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG as compared to running the standard thermal cycling conditions and the TaqMan 2X Universal PCR Master Mix If you encounter poor performance see Troubleshooting on page 48 When performing multiplex applications when more than one target is amplified in a single tube it may be necessary to perform some assay reoptimization Before performing any multiplex applications see the troubleshooting information on page 48 for further information Expert Mode Expert mode allows you to select only those filters required for a particular experiment reducing run times to less than 30 minutes Observe the following guidelines and for detailed information refer to Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Using Expert Mode User Bulletin The default thermal cycling protocol for Fast mode has an extension time of 30 seconds This extension time has been tested for TaqMan Gene Expression Assays TaqMan Pre Developed Assay Reagents and Primer Express Software designed assays that are run using default Fast thermal cycling mode conditions Use of extension times below 30 seconds has been shown to affect the
18. a Click to select a detector for example RNase P Ctrl click to select multiple detectors If no detectors are listed in the Detector Manager click New Detector to open the New Detector dialog box For more information about creating new detectors refer to Appendix A on page 73 Click Add gt gt to add the detectors to the plate document then click Next gt Note To remove a detector from the Detectors in Document panel select the detector then click Remove 6 Specify the detectors and tasks for each well a 40 Click a well or group of wells for replicates to select it Click the detector name s to select the detector s for the well Click under the Task column to assign the detector task Enter a quantity for wells that contain standards Click Use The detector task and color are displayed in the selected wells Click Finish The SDS software creates the plate document New Document Wizard Select Detectors Select the detectors you will be using in the document dA I a at Priore creep eee Ser ws 1 TAEA letector Hame Description Reporter Quenche Detectors in Document i CR2 FAM none RNase P FAM none FAM none Add gt gt l Remove FAM none FAM none FAM none FAM none FAM none FAM none FAM none FAM none tite Poaitiv VIC innne Mi lt New Detector 5 b lt Back Next gt Fini
19. a name for the AQ m Thermal Cycler Protocol plate document then click Save Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 c Load the plate into the precision plate Raps Pae holder in the instrument Ensure that the plate is properly aligned in the holder d Click Start After the run a message indicates 1f the run is successful or if errors were encountered 9 Analyze the AQ data as explained in Chapter 6 Analysis Settings Standard Curve m Ct Analysis a Click m Analysis gt Analysis Settings to 86 configure analysis settings Use the Auto Ct option See Configuring Analysis Settings on page 52 Click OK amp Reanalyze or select Analysis gt Analyze to reanalyze the data If necessary manually adjust the baseline and threshold See Adjusting the Baseline and Threshold on page 54 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Detector Auto Ct Manual Ct Threshold 0 40 E f Automatic Baseline banyal Baseline Start cycle Auto End cycle Auto T Use Sy System Calibration OF amp Reanalyze Cancel Apply d Click OK amp Reanalyze or select Analysis gt Analyze to reanalyze the data e View analysis results Conclusion From the standard curve derive the number of copies of the RNase P gene in population 1 and population 2 Appendix
20. creating an AQ plate document 39 example experiment 37 generating data from RQ plates 33 thermal cycling conditions 44 Fast vs standard plates 35 G graph settings 60 guidelines assay development 14 chemical safety xii chemical waste disposal xiv dissociation curve analysis 77 79 generating standard curves 75 preparing RNA 16 H hazard icons xi hazards chemical waste xiv High Capacity cDNA Reverse Transcription Kits 17 IMPORTANT description xi importing plate setup information 25 39 Information Development department contacting viii Instrument tab 31 45 isothermal assay 79 italic text when to use Vil L line width 60 M master mix PCR 20 34 83 RT 18 82 materials 5 melting curves 64 menu commands conventions for describing vii MSDSs referring to xiii MSDSs obtaining 1x N New Detector dialog box 73 no template control 24 38 normalized reporter 4 NTC 24 38 92 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems O omitting samples 66 options display 60 options graph 60 outliers 66 P passive reference 4 PCR end point 2 master mix preparing 20 34 Real Time 2 starting an AQ plate run 32 47 plate AQ See AQ plates plot appearance 60 Primer Express Software 14 primer dimers 78 primers 14 probes 14 74 protocol dissociation 64 Q quencher dye 62 R Rapid Assay Development Guidelines 14 raw dissociatio
21. from the computer used for data collection when the plate was run For more information about system calibration files refer to the Online Help 5 Click OK amp Reanalyze 6 Examine the amplification plot and if necessary manually adjust the baseline and threshold as explained in the following section Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 53 eee l Chapter 6 Analyzing AQ Data PEE Adjusting the Baseline and Threshold Adjusting the Baseline and Threshold Automatic The SDS software Manual Ct calculates baseline and threshold values for a detector Baseline and based on the assumption that the data exhibits the typical amplification curve Threshold Determination Threshold Cycle A typical amplification curve has a e Plateau phase a e Linear phase b e Exponential geometric phase c e Background d e Baseline e Experimental error such as contamination pipetting errors and so on can produce data that deviate significantly from data for typical amplification curves Such atypical data can cause the software algorithm to generate incorrect baseline and threshold values for the associated detector Therefore Applied Biosystems recommends reviewing all baseline and threshold parameter values after analysis of the study data If necessary adjust the values manually as described on page
22. on the same plate as the target sequence Note The arrangement of the reactions samples and assays on the plate should match the arrangement sample names and detectors markers in the plate document used for the run 2 Into each well of the reaction plate add 50 uL of the appropriate PCR master mix Note Prepare reactions containing standards exactly the same way as reactions containing unknowns Use the same primers and probes PCR master mix components and volume but add to each standard mix a known quantity of template such as cDNA or plasmid DNA Prepare all components in a reaction mix prior to adding to the plate wells 3 Seal the reaction plate with an optical adhesive cover 4 Centrifuge the plate briefly Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 21 ii Chapter 4 Running an AQ Plate 7300 or Standard 7500 System DARD Preparing the Reaction Plate 5 Verify that each reaction is positioned in the bottom of the well Correct Position Incorrect Positions m GR1303 U U The reaction is positioned The reaction lies on the An air bubble lies at the correctly in the bottom of side wall because the bottom of the well the well plate was not centrifuged because the plate was not centrifuged with sufficient force or for sufficient time IMPORTANT Ensure all reaction is positioned correctly i
23. one step RT PCR For more information refer to the Real Time PCR Systems Chemistry Guide Note TaqMan Fast Universal PCR Master Mix does not contain AmpErase UNG enzyme In one step RT PCR RT and PCR take place in one buffer system which provides the convenience of a single tube preparation for RT and PCR amplification However you can not use Fast PCR Master Mix or the carryover prevention enzyme AmpErase UNG uracil N glycosylase to perform one step RT PCR For more information about UNG refer to the Real Time PCR Systems Chemistry Guide 12 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 2 Designing an AQ Experiment Selecting One or Two Step RI PCR Two Step RT PCR One Step RT PCR Sample RNA Sample RNA RT Standard PCR zi T Mix Master Mix Master Mix RT Incubation U Aliquot DNA l l RT incubation and PCR amplification U freme Standard PCR Master Mix OR Fast PCR Master Mix PCR Amplification PCR Amplification and Detection and Detection 2 5 hours M Results 2 hours lt 40 minutes Recommended Kits for Two Step RT PCR Chemistry Step Reagent Part Number TaqMan reagents or kits RT High Capacity cDNA Reverse Transcription 4368813 Kit 1000 reactions High Capacity cDNA Reverse Transcription 4368814 Kit 200 reactions High Capacity cDNA Reverse Transcription 4374967 Kit with RNase Inhibitor 1000 react
24. out the Analysis button is enabled and a message indicates whether or not the run is successful All data generated during the run is saved to the AQ plate document that you specified in step 6 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 4 Troubleshooting Troubleshooting Troubleshooting Observation Chapter 5 Running an AQ Plate 7500 Fast System Possible Cause Action High C values poor precision or failed PCR reactions Target is difficult to amplify Insufficient CDNA template is present e Increase the annealing extension time in the thermal cycler protocol e Increase the annealing extension temperature to 62 C Use 10 to 100 ng of cDNA template per 20 uL reaction Quality of cDNA template is poor 1 Quantitate the amount of cDNA template 2 Test the cDNA template for the presence of PCR inhibitors 3 Measure ODz 69 289 gt 1 8 RNA or 1 9 DNA Sample degradation The TaqMan Universal PCR Master Mix 2X was used instead of the TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG Primer dimer formation Prepare fresh cDNA then repeat the experiment Prepare the reactions with the correct Master Mix To ensure optimal results run the reaction plate as soon as possible after completing the reaction setup If you cannot run a reaction plate within 2 hours after completing the reaction se
25. performance of some assays Validate the performance of assays with extension times of less than 30 seconds Applied Biosystems strongly recommends the use of ROX dye to normalize variation caused by pipetting error When using the ROX passive reference dye feature it is important to select both the FAM and ROX dye filters Add extension time if you use more than three filters to allow for data collection processes Note The filters are labeled Filter A through E by default but may be renamed as desired Select Tools gt Filter Configuration to open the Filter Naming window Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 43 Chapter 5 Running an AQ Plate 7500 Fast System Specifying Thermal Cycling Conditions and Starting the Run Default Thermal Ifyou selected the two step RT PCR method for your AQ experiment recommended Cycling you have already completed the RT step At this point in the workflow you are ready to Conditions for PCR amplify cDNA Users of the 7500 Fast System can choose between the standard PCR default and Fast thermal cycling conditions Expert Mode is disabled by default and it is only available with the 7500 Fast mode Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 The default thermal cycling conditions for the PCR step of the procedure shown in the following table should appear on the Instrument
26. sample name Omit Well Passive Reference d If necessary change the setting for the Ad Detector Remove Cee ROX E Passive Reference dye By default the ROX dye is selected e Repeat steps b through d until you have specified sample names and passive reference dyes for all the wells on the plate then click xj to close the Well Inspector 7b TC 7d 7e Note You can change the sample setup information sample name detector task after a run is complete IMPORTANT If your experiment does not use all the wells on a plate do not omit the wells from use at this point You can omit unused wells after the run For information about omitting unused wells refer to the Online Help 8 Verify the information on each well in the Setup tab Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 2 Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Creating an AQ Plate Document Example Experiment The samples being quantitated and the standards were arranged on a single plate Each well was associated with a detector indicated by the colored squares Each well was also assigned a detector task U unknown S standard or N no template control Only one detector named RNase P was defined because only one gene was being quantitated The figure below shows the example AQ plate document after sample names detectors and detector tasks wer
27. select the detector s for the well c Click under the Task column to assign the detector task d Enter a quantity for wells that contain standards e Click Use The detector task and color are displayed in the selected wells f Click Finish The SDS software creates the plate document Chapter 4 Running an AQ Plate 7300 or Standard 7500 System New Document Wizard Select Detectors Select the detectors you will be using in the document a ad J pont Name Description Reporter Quencher FAM TAMRA RNase P Le ee ed f T SSSR Detectors in Document Internal Positiv FAM TAMRA Add gt gt i lt lt Remove New Detector 5 b lt Back Next gt Finish Cancel 6c 6d New Document Wizard Use Use Detector Reporter REE RNase P FAM TAMRA 6b 6a 6e 6f 26 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Creating an AQ Plate Document Enter the sample names Setup instrument YResuis a Click or select View gt Well Inspector Note To enter sample names without using the Well Inspector click drag to select wells then type the sample name 5K Detecti r Reporter Quencher Task Quantity Color al RNase P FAM TAMRA Unknown b Click a well or click drag to select replicate wells c Enter the
28. standards is required for each gene B A Single gene in two populations B Two genes in two populations 6 RNase P Population 1 RNase P Population 1 RNase P Population 2 NTC STD 1250 STD 2500 STD 5000 STD 10000 STD 20000 IL 10 Population 1 IL 10 Population 2 STD 1250 STD 2500 i STD 5000 STD 10000 STD 20000 NTC STD 1250 STD 2500 STD 5000 STD 10000 STD 20000 RNase P Population 2 GR2341 GR2364 10 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 2 Designing an AQ Experiment Selecting the Chemistry Selecting the Chemistry About Applied Biosystems offers two types of chemistries that you can use to detect PCR Chemistries products on real time instruments as explained in the following table Both TaqMan probe based and SYBR Green I dye chemistries can be used for either one or two step RT PCR For more information about these chemistries refer to the Real Time PCR Systems Chemistry Guide PN 4348358 Chemistry Process TaqMan reagents or kits PCR and Detection of cDNA a Assay Components Description i ge pores ones Wan T a inn Mue TaqMan reagent based chemistry uses a fluorogenic probe to ena
29. the exponential phase of the amplification curve Threshold settings above or below the optimum increase the standard deviation of the replicate groups J Report Al__ i t__ RNaseP Unknown 2551 0 040 478897 473219 178016 A2 iA RNaseP Unknown 2557 0 040 4561 29 473249 1286168 As fiA RNaseP Unknown 2548 0 040 4877 24 473249 1286168 A4 i1 RNeseP Unknown 2554 0 040 4681 28 473219 1288516 OOO et Sampie Detector Task Ct Stanev cay Mean a star ow Threshold Set Too Low The threshold is set below the exponential phase of the amplification curve The standard deviation is significantly higher than that for a plot where the threshold is set correctly Drag the threshold bar up into the exponential phase of the curve Report o o em mm 1 1 RNase P Unknown 20 27 0 576 2677 676 1 1 RNase P Unknown 2157 _ 0576 2677 676 14 RNaseP Unknown 20 73 0 576 2677 676 1 1 __ RNaseP Unknown 21 28 0 576 2677 676 Threshold Set Too High The threshold is set above the exponential phase of the amplification curve The standard deviation is significantly higher than that for a plot where the threshold is set correctly Drag the threshold bar down into the exponential phase of the curve Move down Results _Well Sample Detector Task ct_ stadevct aty Stanev aty Filtered m i 1___ RNaseP Unknown 3824 0481 432550 A2 11 RNase P Unknown 3781 0 481 486756 a3 i 1__ RNaseP
30. 00 7500 Fast Systems Example AQ Experiment Description The objective of the example AQ experiment is to determine the copy number of the RNase P gene in individuals from two populations The experiment is designed for singleplex PCR and primers and probes are designed using Primer Express Software A set of standards is generated by making serial dilutions from a sample of known quantity Data are generated by running a single AQ plate containing both the standard curve and the samples then analyzed using software for the 7300 7500 7500 Fast system Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 81 Appendix E Example AQ Experiment Example AQ Experiment Procedure 1 Design the experiment See Chapter 2 on page 10 a Designate the unknowns prepare the standard curve and determine the number of replicates b Order the reagents for TaqMan probe based chemistry or design primers and probes using Primer Express Software 2 Isolate total RNA See Chapter 3 on page 16 3 Use the High Capacity cDNA Reverse Transcription Kit to generate cDNA from total RNA See Chapter 3 on page 17 a Prepare the reverse transcription RT master mix as indicated in the table to the right Additional guidelines are provided in the High Capacity cDNA Reverse Transcription Kits Protocol PN 4375575 Neer CHEMICAL HAZARD 10 x Reverse Transcription Buffer may cause e
31. 00 7500 Fast Systems 41 Chapter 5 Running an AQ Plate 7500 Fast System Creating an AQ Plate Document Example Experiment The samples being quantitated and the standards were arranged on a single plate Each well was associated with a detector indicated by the colored squares Each well was also assigned a detector task U unknown S Standard or N no template control Only one detector named RNase P was defined because only one gene was being quantitated The figure below shows the example AQ plate document after sample names detectors and detector tasks were assigned for each well Sample Name Detector Task and Color AJ 5 AlLAtae Nira nir ic I y O tU U 2 42 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 5 Running an AQ Plate 7500 Fast System Specifying Thermal Cycling Conditions and Starting the Run Specifying Thermal Cycling Conditions and Starting the Run Running Assays Using Fast Thermal Cycling Conditions The performance of Fast thermal cycling and the TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG has been verified for quantitative applications only and not for endpoint applications such as allelic discrimination The performance of Applied Biosystems TaqMan Gene Expression Assays and Custom TaqMan Gene Expression Assays has been verified using the default Fast thermal cycling conditions and the
32. 7300 7500 7500 Fast Real Time PCR System Allelic Discrimination Getting Started Guide PN 4347822 e Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Plus Minus Getting Started Guide PN 4347821 e Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Relative Quantitation Using Comparative Cr Getting Started Guide PN 4347824 e Applied Biosystems 7300 7500 7500 Fast Real Time PCR System User Guide for the 21 CFR Part 11 Module in SDS Software v1 4 PN 4374432 e Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide PN 4347828 e Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Site Preparation Guide PN 4347823 e Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Performing Fast Gene Quantitation Quick Reference Card PN 4362285 e Applied Biosystems 7500 Fast Real Time PCR System Using Expert Mode User Bulletin PN 4367499 e Applied Biosystems Real Time PCR Systems Computer Setup Guide PN 4365367 e Applied Biosystems Real Time PCR Systems Chemistry Guide PN 4348358 e TaqMan Universal PCR Master Mix Protocol PN 4351891 e Applied Biosystems High Capacity cDNA Reverse Transcription Kits Protocol PN 4375575 Accessing the Access the Online Help system by clicking in the toolbar of the SDS software Online Help window or by selecting Help gt Contents and Index Send Us Your Applied Biosystems welcomes your comments and suggestions for improv
33. 9 6 982 166 and 6 677 151 and corresponding claims in their non US counterparts owned by Applera Corporation 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 AB Design Applied Biosystems ABI PRISM and Primer Express are registered trademarks and Applera FAM MicroAmp MultiScribe ROX TAMRA and Tempus are trademarks of Applera Corporation or its subsidiaries in the U S and or other countries AmpErase AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc SYBR Green is a registered trademark of Molecular Probes Inc Excel Microsoft PowerPoint and Windows are registered trademarks of Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4347825 Rev E 07 2006 About the 7300 7500 7500 Fast System About Absolute About Introduction Quantitation AQ Experiments 2 Designing Specify the Select the Select One or Choose the Probes Chemistry Two Step RI PCR and Primers SAE an AQ Overview Components of an Experiment AQ Experiment Performing Reverse Transcription Guidelines for Convert Preparing RNA Total
34. Applied Biosystems 7300 7500 7500 Fast KS Applied Real Time PCR System Biosystems Absolute Quantitation aon Using Standard Curve s Getting Started Guide Designing an AQ Experiment Performing Reverse Transcription Running an AQ Plate 7300 7500 System Running an AQ Plate 7500 Fast System ee Analyzing AQ data Copyright 2006 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF NOTICE TO PURCHASER The Applied Biosystems 7300 7500 and 7500 Fast Real Time PCR Systems are real time thermal cyclers covered by one or more of US Patents Nos 6 814 934 5 038 852 5 333 675 5 656 493 5 475 610 5 602 756 6 703 236 6 818 437 7 008 78
35. DSS as0 0 deseo od Sew r EEs enw eee wen AERE ewe eee Introduction About the 7300 7500 7500 Fast System 0000 eee eee About Absolute Quantitation 0 0 0 0 0 ces About AQ Experiments 0000 eee eee ees Designing an AQ Experiment OVEIVICW sora 2 ates be ate ead a Seb ave wee ee ave BS ee ere oe r Specifying the Components of an AQ Experiment Selecting the Chemistry 0 0 0 c eee eee eee eee Selecting One or Two Step RT PCR cee ee ee Choosing the Probes and Primers 000 e eee eee eee eee Performing Reverse Transcription Guidelines for Preparing RNA 2 000 cece ees Converting Total RNA to CDNA 2 0 00 cc ees Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 15 16 17 Running an AQ Plate 7300 or Standard 7500 System 19 Before YOU BOI saasa metat oto a te adele asd de db wi ere palm apne aoid oats 20 Preparing the PCR Master Mix 0 00 0 eee eee eee eee 20 Preparing the Reaction Plate 0 0 0c ees 21 Creating an AQ Plate Document 000 ee eee 24 Specifying Thermal Cycling Conditions and Starting the Run 29 Running an AQ Plate 7500 Fast System 33 Belore YOu BEQIN aust ade leh Need woe ae ed re ee dad 34 Preparing the PCR Master Mix 0 000 cee eee eee ees 34 Preparing the Reaction Plate 0 0 ccc ees 35 Creating an AQ
36. E Example AQ Experiment Ct 30 000 29 500 29 000 28 500 28 000 27 500 27 000 26 500 26 000 25 500 3 000 SMa M Show all standard points for selected detector s Detector RNase P X Slope 3 577242 Intercept 41 033016 R2 0 997959 3 200 3 400 3 600 3 800 4 000 4 200 4 400 Log CO Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 87 Appendix E Example AQ Experiment 88 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems References Collins M L Zayati C Detmar J J Daly B Kolberg J A Cha T A Irvine B D Tucker J and M S Urdea 1995 Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays Anal Biochem 226 120 129 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods Enzymol 155 335 350 Saiki R K Scharf S Faloona F et al 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 89 References 90 Absolute Quantitation Using S
37. Make sure that you use the Fast Optical 96 Well Plate on the 7500 Fast Standard Plates System Standard plates will not function properly and may be crushed when using the 96 Well Fast Block Fast Plates Standard Plates PN 4346906 PN 4306737 A Notch at top right Ze XX corner by A12 Notch at top left corner by A1 maximum maximum reaction reaction volume volume 1 Label the reaction plate ensuring that you include a set of standards for every target sequence The standards must be on the same plate as the target sequence Note The arrangement of the reactions samples and assays on the plate should match the arrangement sample names and detectors markers in the plate document used for the run 2 For the 7500 Fast system add 20 uL into each well of the low head space reaction plate of the appropriate PCR master mix Note The reactions containing standards are prepared exactly the same way as reactions containing unknowns You use the same primers and probes PCR master mix components and volume but add to each standard mix a known quantity of template such as cDNA or plasmid DNA All components are prepared in a reaction mix prior to adding to the plate wells 3 Seal the reaction plate with an optical adhesive cover 4 Centrifuge the plate briefly Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 35 Chapter 5 Running an AQ Plate 7500 Fast
38. Mix 2x 25 0 1X Forward primer 5 0 50 to 900 nM Reverse primer 5 0 50 to 900 nM TagMan probe 5 0 50 to 250 nM cDNA sample 5 0 10 to 100 ng Nuclease free water 5 0 Total 50 0 If you design probes and primers using Primer Express Software they must be optimized to work with the universal assay conditions using the volumes listed in the table above Refer to the TaqMan Universal PCR Master Mix Protocol PN 4351891 for primer optimization All TaqMan Gene Expression Assays and Custom TaqMan Gene Expression Assays are formulated so that the final concentration of the primers and probes are within the recommended values Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Preparing the Reaction Plate Preparing the Reaction Plate Standard vs IMPORTANT Make sure that you use the Standard Optical 96 Well Plate on the 7500 Fast Plates Real Time PCR System Fast Optical 96 Well Plates will not fit into the standard block correctly and will result in loss of data Fast Plates Standard Plates PN 4346906 PN 4306737 A Notch at top right Ao corner by A12 Notch at top left corner by A1 ou TTT note Ee ATATAVAVAVATAY 100 uL reaction maximum volume reaction volume 1 Label the reaction plate ensuring that you include a set of standards for every target sequence The standards must be
39. NA is added directly into each master mix Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 83 Appendix E Example AQ Experiment 5 Prepare the reaction plate a Label the reaction plate ensuring that you include a set of standards for every target sequence The standards must be on the RNase P Population 1 same plate as the target sequence sie phase lg E master mix containing cDNA into each well of a standard plate or pipette 20 UL into a Fast plate hese Population 2 c Keep the reaction plate on ice until you are ready to load it into the 7300 7500 7500 Fast system GR2341 Standard Plate Population 1 NTC STD 1250 STD 2500 STD 5000 STD 10000 STD 20000 RNase P Population 2 GR2478 Fast Plate 6 Create an AQ plate document See Creating an AQ Plate Document on page 24 Quick Startup Select the quick startup document mode a Select Start gt All Programs gt Applied Biosystems gt 7300 7500 7500 Fast System gt 7300 7500 7500 Fast System Software g H to start the SDS software a Recent Document s b In the Quick Startup document dialog box select Create New Document Cancel il 84 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems c Select Stand
40. Plate Name field or accept the default then click Next gt Creating an AQ Plate Document Quick Startup document Quick Startup Select the quick startup document mode Create New Document Open Existing Document Recent Document s 1 2 A E Do ae Cancel New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Container Weller Template Blank Document Browse Run Mode Standard 7500 Operator Administrator Comments Plate Name Plate1 Finish Cancel Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 25 Creating an AQ Plate Document 5 Select detectors to add to the plate document a Click to highlight a detector for example RNase P Ctrl click to select multiple detectors If no detectors are listed in the Detector Manager click New Detector to open the New Detector dialog box For more information about creating new detectors refer to Appendix A on page 73 b Click Add gt gt to add the detectors to the plate document then click Next gt Note To remove a detector from the Detectors in Document panel select the detector then click Remove 6 Specify the detectors and tasks for each well a Click on a well or group of wells for replicates to select it b Click on the detector name s to
41. Q See page 24 Plate Document Analyzing AQ Data Specify Thermal Cycling Conditions and Start the Run See page 29 Notes Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 19 Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Before You Begin Check that background and pure dye runs have been performed regularly to ensure optimal performance of the 7300 or Standard 7500 system For more information about calibrating the 7300 7500 system refer to the Online Help and the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Preparing the PCR Master Mix The second step PCR in the two step RT PCR procedure is amplifying the cDNA which you perform using the TaqMan Universal PCR Master Mix Refer to the TaqMan Universal PCR Master Mix Protocol PN 4351891 for details on how to use the reagents The following table lists the universal assay conditions volume and final concentration for using the master mix Neat CHEMICAL HAZARD TaqMan Universal PCR Master Mix 2X No AmpErase UNG may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Reaction Component uL Sample Final Concentration TaqMan Universal PCR Master
42. RNA to cDNA Primer Extended on mRNA P gt Specify AT Prepare the Prepare the Create an AQ Thermal Cycling PCR Master Mix Reaction Plate Plate Document Conditions and Start the Run Running an STANDARD AQ Plate z Standard Before You Begin STANDARD OR Specify Prepare the Prepare the Create an AQ Thermal Cycling PCR Master Mix Reaction Plate Plate Document Conditions and Start the Run Running an AQ Plate Before You Begin Fast Troubleshooting l l Adjust the Analyzing Configure Bane ard Analyze and If Necessary Export AQ E el AQ Data Analysis Settings View the AQ Data Omit Samples Plate Data Threshold Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems iii Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Contents Absolute Quantitation Experiment Workflow Preface How to Use This Guide 0 0000 eee eee How to Obtain More Information 00000 eee ee eee How to Obtain Support 0 0 00 ee ee Safety Safety Alert Words anana ee ee eee ees Good Laboratory Practices 0 0 ee eee General Chemical WarningS 0 00 cece eee eee eee General Biohazard WarningS 00 cece ee eee eee eee General Chemical Waste Hazard Warnings 000000eees OBiainIne NIS
43. System Software Hl to start the SDS software 2 Inthe Quick Startup document dialog box select Create New Document 3 Inthe Assay drop down list of the New Document Wizard select Standard Curve Absolute Quantitation Accept the default settings for Container and Template 96 Well Clear and Blank Document IMPORTANT You cannot use RQ Plate documents for AQ assays and vice versa The information stored in AQ and RQ plate documents is not interchangeable 4 Enter a name in the Plate Name field or accept the default and click Next gt Chapter 5 Running an AQ Plate 7500 Fast System Creating an AQ Plate Document Quick Startup document J Quick Startup Select the quick startup document mode Create Hew Document Open Existing Document Recent Document s Cancel a 4 Ooo o a New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Standard Curve Absolute Quantitation Container 96 Well Clear Template Blank Document Run Mode Fast 7500 Operator Administrator Comments Browse Plate Name Plate1 Finish Cancel Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 39 Chapter 5 Running an AQ Plate 7500 Fast System Creating an AQ Plate Document 5 Select detectors to add to the plate document
44. X Reverse Transcription Buffer 2 0 54 25X dNTPs 0 8 21 6 10X random primers 2 0 54 MultiScribe Reverse Transcriptase 50 1 0 27 U uL Nuclease free water 4 2 113 4 Total 10 270 Each RT reaction is 20 uL see below If you need 5 uL of cDNA at 50 uL total volume for each of 104 PCR reactions per plate see Preparing the PCR Master Mix on page 20 you need 27 RT reactions Extra volume is included to account for pipetting losses as well as extra cDNA for archiving The cDNA plate was then prepared by pipetting in each well e 10 uL ofthe RT master mix e 10 uL of RNA sample Se qeooooo0ooo0odo ee2eee eee 0 0 e e2e2eoee0000 80 EEEE 0 e e2e2eee0 8000 0 e2e e 0 000086 80 The RNA was then converted to cDNA using the thermal profile parameters for two step RT PCR as described in Thermal Profile Parameters for RT on page 17 The cDNA was stored at 20 C until use 18 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 4 ENAEANTENREN gt MEA Running an AQ Plate 7300 or Standard 7500 System Before You Begin See page 20 Introduction Designing an AQ Experiment PCR Master Mix Performing Reverse Transcription Prepare the See page 21 Reaction Plate gt Running an Running an DARD AQ Plate AQ Plate Standard Fast Create an A
45. alyzing AQ Data Chapter 1 Introduction About the 7300 7500 7500 Fast See page 2 System About Absolute See page 2 Quantitation About See page 3 AQ Experiments Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 1 Chapter 1 Introduction About the 7300 7500 7500 Fast System About the 7300 7500 7500 Fast System Description Absolute Quantitation Assay The Applied Biosystems 7300 7500 7500 Fast Real Time PCR System uses fluorescent based PCR chemistries to provide quantitative detection of nucleic acid sequences using real time analysis and qualitative detection of nucleic acid sequences using end point and dissociation curve analysis The Applied Biosystems 7500 Fast Real Time PCR System allows the user to perform high speed thermal cycling giving run times for quantitative real time PCR applications such as relative quantitation in fewer than 40 minutes The 7300 7500 7500 Fast system allows you to perform several assay types using plates in the 96 well format This guide describes the absolute quantitation AQ using standard curve assay For more information about the other assay types refer to the Real Time PCR Systems Chemistry Guide PN 4348358 and the Online Help for the 7300 7500 7500 Fast System Online Help About Absolute Quantitation Definition Real time PCR Assays Using AQ Plate Documents for Plus Minus and AD Assays Absolut
46. ard Curve Absolute Quantitation in the Assay drop down list then click Next gt IMPORTANT You cannot use RQ Plate documents for AQ assays and vice versa The information stored in AQ and RQ plate documents is not interchangeable d Add detectors to the plate document then click Next gt e Specify the detectors and tasks for each well then click Finish T Enter the sample names in the Well Inspector View gt Well Inspector IMPORTANT If your experiment does not use all the wells on a plate do not omit the wells from use at this point You can omit unused wells after the run For more information about omitting unused wells refer to the Online Help The figure on the right shows a completed plate setup with detectors tasks quantities and sample names Appendix E Example AQ Experiment New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Standard Curve Absolute Quantitation Container 96 Well Clear i sst lt lt isi s Template Blank Document o a Browse Run Mode Operator Administrator Comments Plate Name Plate1 Finish Cancel New Document Wizard Select Detectors Select the detectors you will be using in the document Find Passive Reference ROX Detector Name Description Reporter Quencher Detectors in Document none none TAMRA lt
47. are using TaqMan Fast Universal PCR Master Mix you must start the run within 2 hours of preparing the plate The plate can be refrigerated or stored frozen if a run is not started within 2 hours of plate setup The TaqMan Fast Universal PCR Master Mix Protocol PN 4351891 explains how to use the reagents in the kit The following table lists the universal assay conditions volume and final concentration for using the master mix ermar CHEMICAL HAZARD TaqMan Universal PCR Master Mix 2X No AmpErase UNG may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and glove Volume uL Component 20 uL Reaction TaqMan Gene Expression Assay Mix 20X 1 0 e Forward PCR primer 18 uM e Reverse PCR primer 18 uM TaqMan probe 5 uM cDNA template 10 to 100 ng of RNA converted to cDNA RNase 9 0 free water TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG 10 0 Total Volume 20 0 t If you choose to use UNG decrease the volume of cDNA template and RNase free water to 8 8 uL per 20 uL reaction and add 0 2 uL of UNG stock 1 U uL 34 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 5 Running an AQ Plate 7500 Fast System Preparing the Reaction Plate Preparing the Reaction Plate Fast vs IMPORTANT
48. ate 24 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Creating an AQ Plate Document You can enter sample information into a new plate document copy and paste sample information from existing plate documents import sample information from existing plate documents or use a template document to set up new plate documents This section describes setting up new plate documents Refer to the Online Help for information about copying or importing sample information from existing plate documents or using template documents Note The following procedure is illustrated using the example experiment data file see page 7 To create a new AQ plate document 1 Select Start gt All Programs gt Applied Biosystems gt 7300 7500 System gt 7300 7500 System Software H to start the SDS software 2 Inthe Quick Startup document dialog box select Create New Document 3 Inthe Assay drop down list of the New Document Wizard select Standard Curve Absolute Quantitation Accept the default settings for Container and Template 96 Well Clear and Blank Document Choose from Standard 7300 Standard 7500 or 9600 Emulation Run Modes IMPORTANT You cannot use RQ Plate documents for AQ assays and vice versa The information stored in AQ and RQ plate documents is not interchangeable 4 Enter a name in the
49. ate document Optional Click Add Cycle Add Hold or Add Step to add additional isothermal Stages or steps 19 Appendix D Isothermal Assays T Optional To change the default 60 C temperature setting click a temperature field then enter the new temperature 24 7500 Fast System SDS Software Plate1 Isothermal BB Eile View Tools 21CFR11 Instrument Analysis Window Help Usk 468 MAAR Setup Y Instrument Results Audit Trail E Signatures m Instrument Control Temperature Estimated Time Remaining hh mm Sample Heat Sink Block Cover Cycle Disconnect Status Stage Rep Time mm ss Step D Ened State Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 Click a temperature field then enter a new temperature Add Cycle Add Hold Add Step Add Dissociation Stage t Help Settings Sample Volume uL 20 Run Mode Fast 7500 7 Expert Mode i Data Collection Stage 2 Step 1 60 0 0 30 v 8 Optional Click Add Dissociation Stage Note The dissociation stage uses the standard dissociation stage temperatures not the isothermal temperature setting 9 Complete your plate run as directed in Specifying Thermal Cycling Conditions and Starting the Run on page 29 7300 or Standard 7500 System or page 43 7500 Fast System 80 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 75
50. ause there is no control for the efficiency of the reverse transcription step Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 15 Appendix B Guidelines for Generating Standard Curves 76 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Dissociation curve Analysis Overview The 7300 7500 7500 Fast system supports dissociation curve analysis of nucleic acids using SYBR Green I dye The objective of dissociation curve analysis is to determine the melting temperature Tp of a single target nucleic acid sequence within an unknown sample Typical uses of dissociation curves include detection of nonspecific products and primer concentration optimization The process begins by loading a plate with PCR samples and the SYBR Green I dye The plate is loaded into an instrument that has been programmed to slowly elevate the temperature of the plate over several minutes The binding characteristic of the SYBR Green I dye allows the instrument to monitor the hybridization activity of nucleic acids During the run the instrument records the decrease in SYBR Green dye fluorescence resulting from the dissociation of double stranded DNA Results The following figure illustrates a typical dissociation curve from a run to detect nonspecific amplification in cDNA samples 6 0 Main Product 5 0 4 0 3 01 Rn 2 0 0 0 60 90 95 Temp
51. ble detection of a specific PCR product as it accumulates during PCR cycles LEGEND Advantages b Denatured Template and Annealing of Assay Components METET RatidonnPrimer e Increases specificity with a probe Specific SSS Aeverve primer GD Transcriptase hybridization between probe and target on we Bore generates fluorescence signal E O mmr ae e Provides multiplex capability 5 5 Gea Minor Groove e Optimized assays available c Signal Generation arpa cone 3 5 DNA Polymerase e Allows 5 nuclease assay to be carried out eer A TOMO I Probe during PCR jee ee r Forward pima a vs Jugs 3 oe Tn m S A annn ae 3 5 lt Extended Primer SYBR Green I reagents a a a oe Step 1 Reaction setup Ea l The SYBR Green dye Description fluoresces when bound to Uses SYBR Green dye a double stranded DNA double stranded DNA binding dye to detect PCR products as they a Step 2 Denaturation accumulate during PCR cycles O When the DNA is denatured the SYBR Green dye is Advantages released and the fluorescence is drastically reduced e Economical no probe needed y e Yields a melting profile of distinct PCR yields es atep d Polymerization gP y l 8 During extension primers e Increases signal fluorescence as amplification anneal and PCR product i z product length increases O A is generated Limitations ee ss ss ss ee a Binds nonspecifically to all double stra
52. change the parameters For Tab example you can omit samples or manually set the baseline and threshold If you change any parameters you should reanalyze the data File Yiew Tools Instrument Analysis Window Help BEP ATIE Setup Y Instrument Y a a e Ella Plate Y Spectra Y Component Y Amplification Plot _Y Standard Curve Dissociation y Report The Results tab has seven secondary tabs each of which is described below Details are provided in the Online Help To move between views click a tab e To select all 96 wells on a plate click the upper left corner of the plate T EE GEE EE EE Ee ea A 5K 5K 5K 5K 5K 5K 5K u u u u u u u B 5k 5K 5K 5K 5K 5K 5K u u u u u u u E 5K 5K 5K 5K 5K 5K u u u u u u u e To adjust graph settings double click the y or x axis of a plot to display the Graph Settings dialog The adjustable settings depend on which plot you are viewing Graph Settings Real Time Settings Post Run Settings Anis Auto Scale Minimums Anis W Auto Scale C Linear Minimum 0 000 C Linear Minimum Log i Log Masimum i 0 Masimum Asis i autoscaled in RealTime HARIS W Auto Scale Minimum c Masimum Display Options Line Width 2 1 10 Defaults Cancel Apply 60 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 6 Analyzing AQ Data err l Analyzing and Viewing the AQ Data PRE
53. dicating that the threshold has been changed Analyze The Analyze button is enabled after a baseline or threshold setting is changed Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 6 Analyzing AQ Data 7 Adjusting the Baseline and Threshold Example Experiment Setup Y instrument YResuts 7 Plate Y Spectra Y Component Y Amplification Piot Y Standard Curve Y Dissociation Y Repon Delta Rn ys Cycle 1 0e 001 1 0e 000 1 0e 001 4 a a i 1 0e 002 i f j 5 he nd N 106 004 4 35 F Q 11 139 15 17 19 21 239 25 27 29 31 339 35 37 39 Cycle Number Delta Rn A A 1 0e 003 On closer inspection it appears that the baseline and threshold are called correctly and do not need adjustment e The amplification curve begins after the maximum baseline e The threshold is set in the exponential phase of the amplification curve The data was first analyzed using the Auto Ct and Auto Baseline settings resulting in the following amplification plot Notes Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 59 eee l Chapter 6 Analyzing AQ Data PRE Analyzing and Viewing the AQ Data Analyzing and Viewing the AQ Data About the Results In the Results tab you can view the results of the run and
54. ditional comments for the detector Click OK to save the detector and return to the Detector Manager Repeat steps 2 through 8 for the remaining detectors 10 In the Detector Manager click Done when you finish adding detectors Note TaqMan Gene Expression Assays are shipped with an assay information file AIF This text based file contains information about the assays that you ordered including the Applied Biosystems Assay ID number well location of each assay primer concentration and primer sequence The file also indicates the reporter dyes and quenchers if applicable that are used for each assay When creating detectors you use the reporter dye and quencher information and optionally the gene name or symbol for the sample name You can view the contents of AIFs in a spreadsheet program such as Microsoft Excel software Sample Experiment In the example AQ experiment a single detector was created for the single target being quantitated in the assay The detector was named RNase P and assigned a blue color Following conventions the probe was a TaqMan MGB probe labeled with FAM dye TaqMan MGB probes possess a nonfluorescent quencher In AQ experiments where two or more targets are being quantitated a detector is created for each target 14 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Guidelines for Generating Standard Curves Absolute quantitation u
55. e Transcription Kits Protocol PN 4375575 contains additional guidelines for preparing the RNA template The High Capacity cDNA Reverse Transcription Kits are optimized to convert up to 2 ug of total RNA to cDNA per 20 UL reaction Convert enough total RNA so that the final concentration of total RNA converted to cDNA is 10 to 100 ng in 5 uL for each 50 uL PCR reaction Note If you suspect that the RNA contains RNase activity add RNase Inhibitor to the reverse transcription reaction at a final concentration of 1 0 U UL 16 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 3 Performing Reverse Transcription Converting Total RNA to cDNA Converting Total RNA to cDNA Using the High Capacity CDNA Reverse Transcription Kits Thermal Profile Parameters for RT Use the High Capacity cDNA Reverse Transcription Kit to perform the first step RT in the two step RT PCR method Follow the manual method for converting total RNA into cDNA as specified in the High Capacity cDNA Reverse Transcription Kits Protocol PN 4375575 IMPORTANT The protocol is not shipped with the High Capacity cDNA Reverse Transcription Kit Download the protocol from http docs appliedbiosystems com search taf To search for the document select ABI PRISM 6100 Nucleic Acid PrepStation in the Product list box then click Search at the bottom of the page The protocol is listed under the Protoco
56. e assigned for each well Sample Name Detector Task and Color 28 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Specifying Thermal Cycling Conditions and Starting the Run Specifying Thermal Cycling Conditions and Starting the Run Default Thermal Ifyou selected the two step RT PCR method for your AQ experiment recommended Cycling you have already completed the RT step At this point in the workflow you are ready to Conditions for PCR amplify cDNA PCR Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 Stage 3 Reps Reps Reps 40 eso Add Cycle Add Hold Add Step Add Dissociation Stage Delete Help Settings Sample Yolume uL 50 Run Mode Standard 7500 hi Data Collection Stage 3 Step 2 60 0 1 00 ka The default thermal cycling conditions for the PCR step of the procedure shown in the following table Times and Temperatures Two step RT PCR should appear on the Instrument tab Times and Temperatures Two step RT PCR HOLD HOLD 1 RT Step 10 min 25 C 120 min 37 C HOLD 5 sec 85 C Initial Steps PCR Each of 40 cycles Melt AmpErase UNG AmpliTaq Gold DNA 2 PCR Step Activation Polymerase Activation HOLD HOLD Anneal Extend CYCLE 15 sec 95 C 1 m
57. e primer probe sets go to http www allgenes com Custom TaqMan Gene Expression Assays Designs synthesizes formulates and delivers quality controlled primer and probe sets Use this service if the primer probe set you need is not currently available To place an order contact your Applied Biosystems representative Primer Express Software Helps you design primers and probes for your own quantitation assays For more information about using this software refer to the Primer Express Software v3 0 Getting Started Guide PN 4362460 Applied Biosystems provides assay design guidelines that have been developed specifically for quantitation assays When followed these guidelines provide a reliable system for assay design and optimization For information about the assay design guidelines refer to the Real Time PCR Systems Chemistry Guide Example Experiment Primers and probes for RNase P were designed using Primer Express Software 14 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 3 Performing Reverse Transcription Guidelines for See page 16 Preparing RNA Introduction Designing an AQ Experiment Performing Reverse Transcription Running an Running an ARD AQ Plate RAST AQ Plate Standard Fast Analyzing AQ Data Convert See page 17 Total RNA to cDNA Note
58. e quantitation AQ is the process that determines the absolute quantity of a single nucleic acid target sequence within an unknown sample AQ is performed using real time PCR In Real Time PCR you monitor the progress of the PCR as it occurs Data are collected throughout the PCR process rather than at the end of the PCR process end point PCR In Real Time PCR reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than by the amount of target accumulated at the end of PCR Although Plus Minus and Allelic Discrimination AD assays are end point assays Applied Biosystems recommends that you use the 7300 7500 7500 Fast system to perform amplification and view the real time PCR results In the event that an experiment fails you can study the amplification plots to help determine the cause of the failure Use AQ Plate documents to store real time data for Plus Minus and AD assays AQ Plate documents used for troubleshooting Plus Minus and AD assays do not require standard curves 2 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 1 Introduction About AQ Experiments Fx About AQ Experiments AQ Experiment In this document the term AQ experiment refers to the entire AQ assay process Workflow _ beginning with generating cDNA from RNA reverse transcription through analyzing AQ data The AQ experiment workflow
59. eceive 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 https docs appliedbiosystems com msdssearch html 2 Inthe Search field type in the chemical name 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 document to a destination that you choose 4 To have a copy of a document sent by fax or e mail a Select Fax or Email to the left of the document title in the Search Results page b Click RETRIEVE DOCUMENTS at the end of the document list c After you enter the required information click View Deliver Selected Documents Now XIV Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Running an ARD AQ Plate Standard Introduction Designing 1 an AQ Experiment Performing Reverse Transcription Running an AQ Plate Fast Notes An
60. efer to the Online Help for information about the export file types 2 Enter a file name for the export file 3 Click Save To export data for selected wells and or report columns to a spreadsheet application 1 Select File gt Export gt Results 2 Enter a file name for the export file 3 Click Save The Export Settings dialog box opens 70 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 4 Optional Select export settings e Export only selected wells e Apply Report Settings for Data Columns to export the columns selected in the Report Settings dialog box see Report on page 65 5 Click OK To export graphs to PowerPoint 1 Select Tools gt Graph Export gt All to PowerPoint or right click any graph or plate then select Export All To PowerPoint The All to PowerPoint option exports screenshots from all tabs except the Results gt Report tab of the active file Note To export only the current view select Tools gt Graph Export gt To PowerPoint in any view or right click any graph or plate then select Export To PowerPoint 2 When prompted click OK to export to PowerPoint PowerPoint opens and displays your presentation Note Title and document information slides are automatically added to your presentation 3 Optional In PowerPoint modify your presentation 4 In PowerPoint click al Save to save your presentation
61. er dye fluorescence R as a function of cycle You can use this plot to identify and examine irregular amplification mY nature resus Y Plate Y Spectra Y Component Y Amplification Plot Y Standard Cune Y Rn vs Cycle Data An vs Cycle v Detector RNaseP Line Color WellColr Analysis Settings C Auto Ct Manual Ct Threshold 0 728583 Auto Baseline Rn Manual Baseline Start cycle 13 End cycle 115 2 i AAEE ESAE BIIN doe acd i EATS N Ha ose N ead E BARS A EOE A yc DEAR A od SSC A SESS A eee LAI NAOR _ Cycle Number For more onon about R pra to ihe Real Time PCR Systems Chemistry Guide 62 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 6 Analyzing AQ Data Analyzing and Viewing the AQ Data FRE ARn vs Cycle Log View Displays dye fluorescence AR as a function of cycle number You can use this plot to identify and examine irregular amplification and to manually set the threshold and baseline values for the run Setup Instrument Results Plate Amplification Plot Standard Curve Delta Rn vs Cycle 1 0e 001 __ Data BEIT eee Detector RNase P v Line Color well Color 1 0e 000 Analysis Settings C Auto Ct Manual Ct Threshold 0 728583 C Auto Baseline 1 0e 001 Delta Rn 1 0e 002 aye Manual Baseline Start cycle 3 End cycle
62. er path where you would like to save the JPEG files this dialog box C Applied Biosystems SDS Documents Browse Image Options Note The screen may flicker while the system is generating and saving the JPEG files If it happens wait until the flickering stops and the status in the lower right corner of the main 2 gt Click OK Normal Resolution Screen window says Export complete C High Resolution Printer Lv 72 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Before you can use a plate document to run a plate you need to create and apply detectors for all samples on the plate A detector is a virtual representation of a gene or allele specific nucleic acid probe reagent used for analyses performed on instruments To create a detector 1 Select Tools gt Detector Manager Note A plate document any type must be open before you can access the Tools menu 2 Inthe Detector Manager select File gt New 3 Inthe New Detector dialog box enter a name for the detector IMPORTANT The name of the detector must be unique and should reflect the target locus of the assay such as GAPDH or RNase P Do not use the same name for multiple detectors 4 Optionally click the Description field then enter a brief description of the detector Appendix A Creating Detectors Detector Manager Detector List Find al Last Mod
63. erature C Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems a Appendix C Dissociation curve Analysis Viewing Dissociation curve Data Designing Dissociation curve Analysis Experiments Chemistry Kits for Dissociation curve Analysis The dissociation curve plot displays the dual amplification peaks typical of primer dimer formation The amplification from the specific product is displayed with a T of 82 C while the primer dimer product has a characteristically lower T of 75 C To view dissociation curve data select the Dissociation tab then in the Data Type field select e Derivative Displays a plot of the first derivative of the rate of change in fluorescence as a function of temperature e Raw Displays a plot of fluorescence as a function of temperature Setup Y Instrument Y Plate Amplification Flot Standard Curve Y Dissociation Y Report Dissociation Cure 0 16 Data Type TEE i Derivative r Detector SYBERA z 0 12 0 10 0 06 0 06 Der ative 0 04 0 02 0 00 0 02 aji T5 g5 Temperature C The Online Help provides information about using the 7300 7500 7500 Fast system to perform dissociation curve analysis For a detailed explanation of the SYBR Green I double stranded DNA binding dye chemistry refer to e SYBR Green PCR and RT PCR Reagents Protocol PN 4304965 e SYBR Green PCR Master Mix Pr
64. fi 5 oe Woe amp rf El a ae Ue ale SJE Al st i aie ee gh Si gk Sey EEI __ Cycle Number Ct vs Well Position View Displays threshold cycle Cy as a function of well position You can use this plot to locate outliers in detector data sets see Omitting Samples on page 66 for more information Setup Instrument Results f Plate Y Spectra Y Component Y Amplification Plot Standard Curve Ct vs Well Position Data EEEN AEELEIE 7 Detector RNase P hd Line Color S ig Analysis Settings ee apla a ee p g a E EE G a o a e E E Daa Bman eg ee Ba 1 D E g en C Auto Ct LLLI Manual Ct Ct Threshold fo 728583 C Auto Baseline Manual Baseline Start cycle 13 End cycle 115 aa 0 1 4 7 101316192225 28 31 3437 404346 49525558 61 6467 7073767982858891 94 Well Position Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 63 64 Standard Curve Dissociation Chapter 6 Analyzing AQ Data Analyzing and Viewing the AQ Data Displays the standard curve for samples designated as standards The SDS software calculates the quantity of unknown target sequence from the standard curve for the detector for that target sequence Setup Instrument Results Plate Amplification Plot Y Standard Curve Dissociation Y Report Standard Curve 30 000 alae 29 500
65. gh an example experiment The example experiment represents a typical AQ experiment setup that you can use as a quick start procedure to familiarize yourself with the AQ workflow Detailed steps in the AQ workflow are described in the subsequent chapters of this guide Included in these chapters are Example Experiment boxes that provide details for some of the related steps in the example experiment Refer to Appendix E Example AQ Experiment on page 81 for more information To view the example experiment data file in the SDS software 1 Select File gt Open 2 Navigate to Applied Biosystems SDS Documents Example Data Files EXAMPLE _ AQ sds then click Open Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems T Chapter 1 Introduction About AQ Experiments 8 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 2 Designing an AQ Experiment See page 10 Overview Introduction Specify the Components of an AQ Experiment See page 10 Designing an AQ Experiment Performing Reverse Transcription Select the See page 11 Chemistry Running an Running an ARD AQ Plate RAST AQ Plate Standard Fast Select One or See page 12 Two Step RI PCR Analyzing AQ Data Choose the Probes See page 14 and Primers
66. gloves JN DANa ELECTRICAL HAZARD Failure to ground the instrument properly can lead to an electrical shock Ground the instrument according to the provided instructions Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems XI Safety Good Laboratory Practices Good Laboratory Practices PCR Good Laboratory Practices PCR assays require special laboratory practices to avoid false positive amplifications Kwok and Higuchi 1989 The high throughput and repetition of these assays can lead to amplification of a single DNA molecule Saiki et al 1985 Mullis and Faloona 1987 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 Change gloves whenever you suspect that they are contaminated Maintain separate areas dedicated equipment and supplies for Sample preparation and PCR setup PCR amplification and post PCR analysis Never bring amplified PCR products into the PCR setup area Open and close all sample tubes and reaction plates carefully Do not splash or spray PCR samples Keep reactions and components sealed as much as possible Use positive displacement pipettes or aerosol resistant pipette tips Clean lab benches and equipment periodically with freshly diluted 10 bleach solution Bibliography Kwok S and Higuchi R 1989 Avoiding false posit
67. has several steps shown in the figure on page 111 AQ assays use a standard curve to calculate the quantity of an unknown target sequence The results of AQ experiments are reported in the same units of measure as the standard curve The 7300 7500 7500 Fast system stores Real Time PCR data collected from the reaction plate in an AQ Plate document Each run consists of a single plate The 7300 7500 7500 Fast system provides several views for analyzing data Plated Reactions 7300 7500 7500 Fast Instrument SDS Software AQ Plate Document Analyze Results Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 3 Terms Used in Quantitation Analysis Chapter 1 Introduction About AQ Experiments Term Definition Baseline The initial cycles of PCR in which there is little change in fluorescence signal Threshold A level of delta R automatically determined by the software or manually set used for C 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 curve The threshold is the line whose intersection with the Amplification plot defines the Cy Threshold cycle Cy The fractional cycle number at which the fluorescence passes the threshold No template control NTC A sample that does not contain template It
68. iability across the reaction ROX dye was not selected as the Select ROX dye as the passive plate passive reference when the plate reference when you set up the plate document was set up document Evaporation Make sure that the reaction plate is sealed completely especially around the edges High variability across replicates Reaction mix was not mixed well Mix the reaction mix gently by inversion then centrifuge briefly before aliquoting to the reaction plate Troubleshooting Multiplex Applications IMPORTANT Due to the challenging nature of multiplex applications and the complexity that can be encountered it is impossible to guarantee assay performance However the recommendations listed below should be helpful when running multiplex applications using Fast thermal cycling conditions and the TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG Perform the recommendations in the order listed When running multiplex applications 1 Increase the annealing extension temperature to 62 C 2 If you do not obtain the expected performance by increasing the annealing extension temperature to 62 C increase the annealing extension time in the thermal cycling protocol by 5 seconds to 35 seconds 3 If you do not obtain acceptable performance by increasing both the annealing extension temperature and time assay reoptimization may be required Refer to the Real Time PCR Systems Chemistry Guide PN 4348358 for
69. in 60 C 2 min 50 C 10 min 95 C Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 29 Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Specifying Thermal Cycling Conditions and Starting the Run Thermal Cycling If you select the one step RT PCR method cDNA generation and amplification take Conditions for place simultaneously at this point in the workflow One Step RT PCR Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 Stage 3 Reps Reps Reps Add Cycle Add Hold Add Step Add Dissociation Stage Help Settings Sample Volume pL 50 Run Mode Standard 7500 v Data Collection Stage 3 Step 2 60 0 1 00 v The following table Times and Temperatures One step RT PCR shows the thermal cycling conditions for one step RT PCR experiments Times and Temperatures One step RT PCR Initial Steps PCR Each of 40 Cycles Reverse Transcription AmpliTag Gold DNA Melt Anneal Extend Polymerase Activation HOLD HOLD CYCLE 30 min 48 C 10 min 95 C 15 sec 95 C 1 min 60 C 30 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Specifying Thermal Cycling Conditions and Starting the Run To specify thermal cycling conditions a
70. ing its user Comments documents You can e mail your comments to techpubs appliedbiosystems com viii Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Preface How to Obtain Support How to Obtain 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 Support At the Support page you can Obtain worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities 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 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Ix Preface How to Obtain Support X Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Safety Safety Alert Words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observatio
71. ions High Capacity cDNA Reverse Transcription 4374966 Kit with RNase Inhibitor 200 reactions PCR TaqMan Universal PCR Master Mix 4304437 TaqMan Fast Universal PCR Master Mix 2X 4352042 No AmpErase UNG Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 13 Chapter 2 Designing an AQ Experiment Choosing the Probes and Primers Recommended Kits for Two Step RT PCR Chemistry Step Reagent Part Number SYBR Green reagents or kits RT High Capacity cDNA Reverse Transcription 4368813 Kit 1000 reactions High Capacity cDNA Reverse Transcription 4368814 Kit 200 reactions High Capacity cDNA Reverse Transcription 4374967 Kit with RNase Inhibitor 1000 reactions High Capacity cDNA Reverse Transcription 4374966 Kit with RNase Inhibitor 200 reactions PCR SYBR Green PCR Master Mix 4309155 Power SYBR Green PCR Master Mix 4367659 RT and PCR SYBR Green RT PCR Reagents 4310179 Example Experiment Used two step RI PCR with the TaqMan reagents and kits indicated in the table above Choosing the Probes and Primers Choose probe and primer sets for your target sequences Applied Biosystems provides three options for choosing primers and probes TaqMan Gene Expression Assays Provide you with optimized ready to use TaqMan assays 5 nuclease for human mouse or rat transcripts For information on availabl
72. is used to verify amplification quality Nucleic acid target also called template Nucleotide sequence that you want to detect and quantitate Passive reference A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis Normalization is necessary to correct for fluorescence fluctuations caused by changes in concentration or in volume Reporter dye The dye attached to the 5 end of a TaqMan probe The dye provides a fluorescence signal that indicates specific amplification Normalized reporter Rn The ratio of the fluorescence emission intensity of the reporter dye to the fluorescence emission intensity of the passive reference dye Delta R AR The magnitude of the signal generated by the specified set of PCR conditions AR R baseline Standard A sample of known quantity used to construct a standard curve Unknown sample A sample containing an unknown quantity of template that you want to characterize Threshold The figure below shows a representative amplification plot and includes some of the terms defined above No Template Control 0 5 10 15 20 25 30 35 40 Cycle Number 4 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Required User Supplied Materials Chapter 1 Introduction About AQ Experiments 3 Item
73. ives with PCR Nature 339 237 238 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods Enzymol 155 335 350 Saiki R K Scharf S Faloona F et al 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 General Chemical Warnings Chemical Hazard Neo CHEMICAL HAZARD Some of the chemicals used with Warning Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Chemical Safety To minimize the hazards of chemicals xii Guidelines e 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 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 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Safety General Biohazard Warnings e 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 e Check regularly for chemical leaks or spills If a leak
74. ls heading The High Capacity cDNA Reverse Transcription Kits use the following thermal profile parameters for the RT step Step Type Time Temperature HOLD 10 min 25 C HOLD 120 min 37 C HOLD 5 sec 85 C Note If you are using a standalone thermal cycler you can add an additional 4 C HOLD step For more information see the Applied Biosystems High Capacity cDNA Reverse Transcription Kits Protocol PN 4375575 Note Thermal cycling conditions for one step RT PCR are described on page 30 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 17 Chapter 3 Performing Reverse Transcription Converting Total RNA to cDNA Storing cDNA After cDNA conversion store all cDNA samples at 15 to 25 C To minimize repeated freeze thaw cycles of cDNA store cDNA samples in aliquots Nore CHEMICAL HAZARD 10 x Reverse Transcription Buffer may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Example Experiment Standard Plate For the example experiment total RNA was extracted from blood RNA concentration was determined using Avgo The RT master mix was prepared as follows using guidelines from the High Capacity cDNA Reverse Transcription Kits Protocol PN 4375575 Component uL Reaction uL 27 Reactions 10
75. more information Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 49 Chapter 5 Running an AQ Plate 7500 Fast System Troubleshooting 50 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Introduction Designing an AQ Experiment Performing Reverse Transcription Running an Running an AQ Plate Standard Fast Analyzing AQ Data Notes Chapter 6 Analyzing AQ Data Configure See page 52 Analysis Settings Adjust the Baseline and Threshold See page 54 Analyze and See page 60 View the AQ Data If Necessary l See page 66 Omit Samples Export AQ See page 70 Plate Data Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 51 eee l Chapter 6 Analyzing AQ Data PEE Configuring Analysis Settings Configuring Analysis Settings Before you can analyze the data you must specify parameter values for the analysis Unless you have already determined the optimal baseline and threshold settings for your experiment use the automatic baseline and threshold feature of the SDS software Auto Ct If the baseline and threshold were called correctly for each well you can proceed to view the results Otherwise you must manually set the baseline a
76. n data 78 reagent configurations 12 reference passive 4 replicates 10 reporter dye 4 62 results 60 reverse transcription guidelines for preparing RNA 16 High Capacity cDNA Reverse Transcription Kits 17 thermal cycling parameters 17 Rn vs Cycle view 62 Rn See normalized reporter RNA converting to cDNA 17 guidelines for preparing 16 starting concentration 16 ROX dye 27 41 62 RQ plates detectors creating 73 RT PCR kits for two step 13 one step 12 30 Index thermal cycling conditions 29 44 two step 12 S safety biological hazards xiii chemical waste xiv settings graph 60 Setup tab 27 41 standard 24 38 standard curves 10 75 standard deviation effect of threshold on 56 standard vs fast plates 21 starting an AQ plate run 32 47 SYBR Green I dye chemistry 11 y TAMRA dye 62 74 TaqMan Assay chemistry 11 TaqMan Gene Expression Assays 14 TaqMan MGB probes 62 74 TaqMan Universal PCR Master Mix 20 34 Technical Support contacting ix template documents 25 39 text conventions vii thermal cycling conditions default for PCR 29 44 forRT 17 one step RT PCR 30 specifying 31 43 45 threshold adjusting 54 examples 56 threshold cycle definition 4 setting for AQ studies 52 training information on ix U unknown 24 38 uracil N glycosylase 12 user attention words described vii V viewing AQ data 60 W WARNING description vii x1 Absolute Quantitation Using Standard Curve Ge
77. n 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 07tl 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 Nem Indicates a potentially hazardous situation that if not avoided could result in death or serious injury N py lela 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 Important 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 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 Nee 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
78. n the bottom of the well before starting a run Failure to do so will impact the quality of data 6 Keep the reaction plate on ice until you are ready to load it into the 7300 7500 system 22 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Preparing the Reaction Plate Example Experiment The PCR master mixes were prepared according to the universal assay conditions pac ton COmponeni peneachon nea nei Ma Ca aion TaqMan Universal PCR Master Mix 2X 25 0 125 0 925 0 1X Forward primer 5 0 25 0 185 0 50 to 900 nM Reverse primer 5 0 25 0 185 0 50 to 900 nM TaqMan probe 5 0 25 0 185 0 50 to 250 nM cDNA sample or template for standards 5 0 25 0 185 0 10 to 100 ng Nuclease free water 5 0 25 0 185 0 Total 50 0 250 0 1850 0 One master mix was prepared for each of the six standards 4 replicates plus extra volume for pipetting losses One master mix was prepared for each of the two populations being studied 36 samples plus extra volume for pipetting losses Unknowns target sequences being quantitated and standards were arranged on a plate 50 uL of the appropriate PCR RNase P Population 1 STD 1250 STD 2500 STD 5000 STD 10000 STD 20000 RNase P Population 2 GR2341 master mix containing cDNA
79. nd start the run 1 Select the Instrument tab Instrument Control Temperature By default the standard PCR conditions for the i Estimated Time Remaining hh mm pies ee PCR step of the two step RT PCR method are lt Cycle Status Stage Rep di splayed Time mm ss Step State PA Veri fy tha t Thermal Cycler pis Thermal Profile Auto Increment Ramp Rate e Ifyou are using two step RT PCR The ei oe ee default PCR thermal cycling conditions are set Note If you are using one step RT PCR use the thermal cycling parameters shown in Add Cycle Add Hold Add Ste Add Dissociation Stage Hel Thermal Cycling Conditions for One Step a ae Es ee ea ee RT PCR on page 30 Sample Volume uL 50 Run Mode Standard 7500 gt Data Collection Stage 3 Step 2 60 0 1 00 v e Sample Volume is 50 uL e Verify desired Run Mode Note If you are using SYBR Green I reagent chemistry and you want to determine 1f there is contamination or if you want to determine the dissociation temperature of your amplicons click Add Dissociation Stage The dissociation stage includes a post dissociation cooling step Refer to the Online Help for more information Note In the 7300 instrument the 9600 Emulation feature is not available Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 31 Chapter 4 Running an AQ Plate 7300 or Standard 7500 System Specif
80. nd threshold as explained in Manual Baseline and Threshold Determination on page 54 This section describes how to use the auto Ct feature To configure analysis settings 1 Click Analysis gt Analysis Settings Analysis Settings Standard Curve Ct Analysis 2 Inthe Detectors drop down list select All tees 3 Select Auto Ct The SDS software automatically Auto Ct 3 generates baseline values for each well and oon threshold values for each detector Threshold 0 4016 IMPORTANT After analysis you must verify 2 Start cycle Auto End cycle Auto that the baseline and threshold were called correctly for each well as explained in Adjusting the Baseline and Threshold on 4 page 54 Ok amp Reanalyze Cancel Apply 5 Alternatively select Manual Ct and specify the threshold and baseline manually You can also select Auto baseline and Manual Ct 52 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Configuring Analysis Settings Chapter 6 Analyzing AQ Data FREE 4 Optional Select Use System Calibration to use the calibration files that are stored on the computer you are currently using Note If you do not select Use System Calibration the calibration information stored in your plate document is used This information comes
81. nded DNA 0 0 een pada a A a inte ys ale aa Aae a reen dye binds to the sequences To avoid false positive signals check _ Se e e e i daube ama o Ne for nonspecific product formation using dissociation resulting in a net increase in curve or gel analysis fluorescence detected by the instrument Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 11 Chapter 2 Designing an AQ Experiment Selecting One or Two Step RT PCR Selecting One or Two Step RT PCR When performing real time PCR you have the option of performing reverse transcription RT and PCR in a single reaction one step or in separate reactions two step The reagent configuration you use depends on whether you are performing two step or one step RT PCR e Two step RT PCR is performed in two separate reactions First total RNA is reverse transcribed into cDNA then the cDNA is amplified by PCR This method is useful for detecting multiple transcripts from a single cDNA template or for storing cDNA aliquots for later use AmpErase UNG enzyme can be used to prevent carryover contamination Users of the 7500 Fast system can use TaqMan Fast Universal PCR Master Mix or TaqMan Universal PCR Master Mix for an approximately 40 minute run time or a 2 hour run time respectively IMPORTANT This guide emphasizes AQ experiments that are designed using two step RT PCR but also provides information on
82. odborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx 01 29cfr1910a_01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at http www cdc gov Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems xili Safety General Chemical Waste Hazard Warnings General Chemical Waste Hazard Warnings Chemical Waste NUE HAZARDOUS WASTE Refer to Material Safety Data Sheets and Hazard local regulations for handling and disposal AINTE 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 Obtaining MSDSs 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 r
83. old 4 Set the baseline for the detector a Under Analysis Settings select Manual Baseline b Enter values in the Start cycle and End cycle fields ensuring that the amplification curve growth begins at a cycle after the End Cycle value Note After you change a baseline or threshold setting for a detector the Analyze button nayz is enabled indicating that you must reanalyze the data 5 Set the threshold for the detector 6 08 a Under Analysis Settings select Manual Ct b Drag the threshold setting bar until the threshold is e Above the background e Below the plateau and linear regions of the amplification curve e Within the exponential phase of the amplification curve The SDS software adjusts the threshold value and displays it in the Threshold field after the analysis Repeat steps 3 through 4 to set the baseline and threshold values for all remaining detectors in the study Click Analysis gt Analyze to reanalyze the data using the adjusted baseline and threshold values Delta Rn vs Cycle Data Delta An vs Cycle v Detector RNase P xz Line Color well Color v Analysis Settings C Auto Ct Manual Ct Threshold 0 089244 Auto Baseline Manual Baseline Start cycle 12 End cycle 14 URE fy HE ail E he e AJ a 2 F ayy ER R SE fh ay EE __ Cycle Number Drag the Threshold bar to adjust the threshold The bar turns red in
84. on 40 D Outlier 39 3 Select wells to examine then verify the uniformity of each replicate population by i comparing the groupings of Cy values 25 _ 4 If you identify an outlier locate the associated well 15 a Determine the approximate well position of 10 the outlier from the x axis of the plot 0 1 4 7 1013161922252831 3437 40 43 4649525558 61 6467 7073 7679828588 91 94 Vell Position 66 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems b In the plate grid select a range of wells that includes the approximate well position of the outlier The plot displays only the selected wells c From the plot determine the exact well position of the outlier d Select View gt Well Inspector then select the Omit check box for the appropriate well Chapter 6 Analyzing AQ Data Omitting Samples Ct vs Well Position 42 000 vig D Outlier 35 000 30 000 25 000 a a 5 20 000 15 000 10 000 5 000 0 000 pama DEGEE mmes 42 Outlier is 40 shown in plot Use Detector Reporter quencher Task Quantity Color IY RNaseP FAM none NTC Select l Omit Well E omwei Da Passive Reference check box 1 2 3 4 5 6 7 Si u u u u o u Omitted E u u u u u u well is 0 u u u u 1 N LESZ a crossed out Absolute Quantitation Using Standard Curve Getting Started G
85. or one step RT PCR or EZ RT core reagents d Store the cDNA at 20 C until use 4 Prepare the PCR master mix as indicated in the table to the right See Chapter 4 on page 20 for more information Note The reaction volumes for TagMan Gene Expression Assays and TaqMan Custom Gene Expression Assays are specified in the product insert that accompanies these products Nexen CHEMICAL HAZARD TaqMan Universal PCR Master Mix 2X No AmpErase UNG may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Step Type Time Temperature HOLD 10 min 20 C HOLD 120 min 37 C HOLD 5 sec 85 C PCR Master Mixt Reaction Standard Fast Final Component uL uL Concen Sample Sample tration TaqMan 25 0 10 0 1X Universal PCR Master Mix 2X or TaqMan Fast Universal PCR Master Mix Forward primer 5 0 2 0 50 to 900 nM Reverse primer 5 0 2 0 50 to 900 nM TaqMan probe 5 0 2 0 50 to 250 nM cDNA 5 0 2 0 10 to 100 sample ng Nuclease free 5 0 2 0 water Total 50 0 20 0 For the example experiment eight PCR master mixes were prepared one for each of the two sample populations for 37 reactions and one for each of the six standards for 5 reactions Include extra volume to account for pipetting losses cD
86. or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS e Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal General Warnings Nem CHEMICAL HAZARD Before handling any chemicals refer to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions General Biohazard Warnings General ANTE BIOHAZARD Biological samples such as tissues body fluids Biohazard infectious agents 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 equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following 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 Blo
87. otocol PN 4310251 The following Applied Biosystems kits are available Kit Part Number SYBR Green RT PCR Reagents 4310179 SYBR Green PCR Core Reagents 4304886 SYBR Green PCR Master Mix 4309155 Power SYBR Green PCR Master Mix 4367659 78 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Isothermal Assays The 7300 7500 7500 Fast System supports isothermal assays Creating an 1 Isothermal Assay Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Select Start gt All Programs gt Applied Biosystems 7300 7500 7500 Fast System gt 7300 7500 7500 Fast System Software K to start the SDS Software In the Quick Startup document dialog box select Create New Document In the assay drop down list of the New Document Wizard select Isothermal New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Isothermal Container 96 Well Clear nd Template Blank Document id Browse Run Mode Standard 7500 r Operator Administrator Comments Plate Name Plated Finish Cancel Complete your plate set up as directed in Creating an AQ Plate Document on page 25 7300 or Standard 7500 System or page 39 7500 Fast System Click the Instrument tab to view the Thermal Profile of the new pl
88. p for information on configuring the User Defined columns Setup Y Instrument Plate Spectra Y Component Amplification Plot Standard Curve Y Dissociation Y Report Well Detector Task Ct St 5K RNase P Unknown 28 0 066 A2 5K RNase P Unknown 26 0 066 AS 5K RNase P Unknown 26 0 066 Ag 5K RNase P Unknown 26 0 066 AS 5K RNase P Unknown 26 0 066 AB 5K RNase P Unknown 26 0 066 AT 5K RNase P Unknown 28 0 066 AS 5K RNase P Unknown 28 0 066 Ag 5K RNase P Unknown 28 0 066 410 5K RNase P Unknown 28 0 066 Alt 5K RNase P Unknown 28 0 066 A12 5K RNase P Unknown 26 0 066 51 5K RNase P Unknown 26 0 066 B2 5K RNase P Unknown 26 0 066 B3 5K RNase P Unknown 26 0 066 B4 5K RNase P Unknown 26 0 066 Note To select the column used to sort the data click the column heading to sort in ascending first and alternating clicks or descending alphanumeric order The Report Settings dialog box formats the display of the report and how the report will be printed You have the option see Exporting AQ Plate Data on page 70 to apply these report settings when you export data Refer to the Online Help for more information about this dialog box Standard Curve Report Settings Report Orientation Portrait Landscape Landscape recommended with 9 or more Data Columns Data Columns Graphis to Print in the Report Well ve i Raw Spectra i Amplification Plot Sample Hame Detector f Portrait f Portrait Task Ct StdDev Ct Quantity E W
89. r Both the FAM and ROX dye filters must be turned on in order to excite the ROX passive reference dye in Applied Biosystems Master Mix correctly Note For information on changing Filter names refer to Online Help or the Applied Biosystems 7500 Fast Real Time PCR System Using Expert Mode User Bulletin 6 Select File gt Save As enter a name for the AQ plate document then click Save Optional If you want to use this plate document again save it as a template document Select File gt Save As In the Save in drop down list navigate to Applied Biosystems 7300 7500 7500 Fast System Templates Type the File name then select sdt for Save as type to save the file as a template T Load the plate into the precision plate holder in the instrument Ensure that the plate is properly a ann aligned in the holder PP 99990900 Note The A1 position is in the top left side of the instrument tray The bar code is toward the front of the instrument I OOOOOCOCOOshf 46 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 5 Running an AQ Plate 7500 Fast System Specifying Thermal Cycling Conditions and Starting the Run 8 Click Start As the instrument performs the PCR run it displays real time status information in the Instrument tab and records the fluorescence emissions After the run the status values and buttons are grayed
90. re the Reaction Plate Performing Reverse Transcription Create an AQ See page 38 Plate Document Running an AQ Plate Fast Running an Standard Specify Thermal Cycling Conditions and Start the Run See page 43 Analyzing AQ Data Troubleshooting See page 48 Notes Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 33 Chapter 5 Running an AQ Plate 7500 Fast System Before You Begin Before You Begin Check that background and pure dye runs have been performed regularly to ensure optimal performance of the 7500 Fast system For more information about calibrating the 7500 Fast system refer to the Online Help and the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide Preparing the PCR Master Mix The second step PCR in the two step RT PCR procedure is amplifying the cDNA which you perform using the TagMan Universal PCR Master Mix reagents Users of the 7300 7500 system must use standard TaqMan Universal PCR Master Mix 2X for a 2 hour run time Users of the 7500 Fast System can choose either the TaqMan Universal PCR Master Mix 2X or TaqMan Fast Universal PCR Master Mix 2X for a run time of fewer than 40 minutes For further information on the use of Fast Master Mix refer to TaqMan Fast Universal PCR Master Mix Protocol PN 4351891 IMPORTANT If you
91. s Chapter 1 Introduction About AQ Experiments Item Source Labeled primers and probes from one of the following sources e TaqMan Gene Expression Assays predesigned primers and probes inventoried e Applied Biosystems PN 4331182 non inventoried e Applied Biosystems PN 4351372 e Custom TaqMan Gene Expression Assays service predesigned primers and probes Small Scale 20X 144 x 50 uL e Applied Biosystems PN 4331348 reactions Medium Scale 20X 300 x 50 uL e Applied Biosystems PN 4332078 reactions Large Scale 60X 1160 x 50 uL e Applied Biosystems PN 4332079 reactions e Primer Express Software custom designed primers and probes 4 user license e Applied Biosystems PN 4363991 user license e Applied Biosystems PN 4363993 6700 Reagent Tubes 10 mL Applied Biosystems PN 4305932 Centrifuge with adapter for 96 well plates Major laboratory supplier MLS Gloves MLS Microcentrifuge MLS Microcentrifuge tubes sterile 1 5 mL MLS Nuclease free water MLS Pipette tips with filter plugs MLS Pipettors positive displacement MLS Vortexer MLS 6 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 1 Introduction About AQ Experiments Example AQ Experiment Overview To better illustrate how to design perform and analyze AQ experiments this section guides you throu
92. s Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 15 Chapter 3 Performing Reverse Transcription Guidelines for Preparing RNA Guidelines for Preparing RNA Isolating Total Applied Biosystems supplies several instrument systems chemistries and protocols for RNA RNA isolation from a variety of starting materials such as blood tissue cell cultures Quality of RNA Adjusting the Starting Concentration of Total RNA and plant material System Chemistry or Protocol Source ABI Prism 6100 Nucleic Acid PrepStation Applied Biosystems PN 6100 01 6100 Reagents and Disposables Starter Kit Applied Biosystems PN 4328773 Tempus Blood RNA Tube Applied Biosystems For collection stabilization and isolation of total RNA in PN 4342792 whole blood for gene expression analysis using the 6100 PrepStation Isolation of Total RNA from Whole Blood and from Cells Applied Biosystems Isolated from Whole Blood Protocol PN 4332809 Tempus Blood RNA Tube and Large Volume Applied Biosystems Consumables Protocol PN 4345218 Tissue RNA Isolation Isolation of Total RNA from Plant Applied Biosystems and Animal Tissue Protocol PN 4330252 The total RNA you use for AQ experiments should e Have an Aj oneq greater than 1 9 e Be intact when visualized by gel electrophoresis e Not contain RT or PCR inhibitors The High Capacity cDNA Revers
93. sh Cancel 6c 6d Unknown Standard NTC 6e 6f Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 5 Running an AQ Plate 7500 Fast System Creating an AQ Plate Document T Enter the sample names a Click or select View gt Well Inspector Note To enter sample names without using the Well Inspector click drag to select wells then type the sample name b Click well or click drag to select replicate wells San ple Name 5K Ube Detector Reporter Quencher Al none Unknown d If necessary change the setting for the aiae Passive Reference Passive Reference dye By default the ls _ Pd Detector Remove Close Rox Be ROX dye is selected c Enter the sample name e Repeat steps b through d until you have specified sample names and passive reference dyes for all the wells on the plate then click xj to close the Well Inspector 7b 7c 7d 7e Note You can change the sample setup information sample name detector task after a run is complete IMPORTANT If your experiment does not use all the wells on a plate do not omit the wells from use at this point You can omit unused wells after the run For information about omitting unused wells refer to the Online Help 8 Verify the information on each well in the Setup tab Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 75
94. sing the 7300 7500 7500 Fast system requires that the absolute quantities of the standards be determined by 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 The following critical points must be considered for the proper use of absolute standard curves e The standard DNA or RNA must be a single pure species For example plasmid DNA prepared from E coli is often contaminated with RNA increasing the A measurement and inflating the copy number determined for the plasmid e Accurate pipetting is required because the standard must be diluted over several orders of magnitude Plasmid DNA or in vitro transcribed RNA must be concentrated to measure an accurate A value This concentrated DNA or RNA must be diluted 10 to 10 fold to be at a concentration similar to the target in biological samples e The stability of the diluted standards must be considered 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 is provided by Collins et al 1995 who report on the steps they used in developing an absolute RNA standard for viral quantitation e Generally it is not possible to use DNA as a standard for absolute quantitation of RNA bec
95. tab Fast Default Times and Temperatures Two step RT PCR HOLD HOLD HOLD 1 RT Step 10 min 25 C 120 min 37 C 5 sec 85 C Fast Thermal Cycling Conditions Fast 7500 users only 2 PCR Step Enzyme Activation Melt Anneal Extend Fast Conditions 20 sec 95 C 3 sec 95 C 30 sec 60 C Expert Mode 20 sec 95 C 3 sec 95 C 20 sec 60 C Conditions 44 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems To specify thermal cycling conditions and start the run 1 Select the Instrument tab By default the standard PCR conditions for the PCR step of the two step RT PCR method are displayed 2 Verify that e Ifyou are using two step RT PCR The default PCR thermal cycling conditions are set Chapter 5 Running an AQ Plate 7500 Fast System Specifying Thermal Cycling Conditions and Starting the Run Note If you are using one step RT PCR set the thermal cycling parameters as shown in Default Thermal Cycling Conditions for PCR on page 44 e Sample Volume is 20 uL for 7500 Fast e Fast 7500 is selected as the Run Mode Note If you are using SYBR Green I dye chemistry and you want to determine if there is contamination or if you want to determine the dissociation temperature of the amplicons click Add Dissociation Stage The dissociation stage includes a post dissociation cooling step
96. tandard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Numerics 5 nuclease assay 11 A absolute quantitation AQ 2 absolute quantitation AQ experiments See AQ experiments AIF See assay information files AmpErase UNG 12 amplification curve 54 amplification plots representative 4 types 60 analysis settings configuring 52 appearance of graphs 60 Applied Biosystems contacting ix customer feedback on documentation viii Information Development department viii Technical Support ix AQ experiments analyzing AQ data 60 chemistries 11 components 10 example experiment 81 probes and primers 14 reagent configuration 11 requirements 10 terms 4 workflow 3 AQ plates Amplification Plots 60 AQ Plate documents 24 38 datatypes 70 exporting data 70 omitting samples from 66 preparing master mix 20 34 preparing reaction plate 21 35 results 60 starting a run 32 47 troubleshooting 48 assay design guidelines 14 assay information files 74 assay isothermal 79 assumptions for using this guide vil Index autoscaling options 60 B baseline 4 adjusting 54 examples 55 biological hazard guidelines xiii bold text when to use vii C calibrating the 7300 7500 instrument 20 calibrating the 7500 Fast instrument 34 CAUTION description vii xi cDNA generating 17 storing 18 chemical safety guidelines xii chemical waste hazards xiv chemical waste disposal guidelines xiv chemistries 11 conven
97. tions bold text vii for describing menu commands vii IMPORTANTS vii in this guide vii italic text vii Notes vii user attention words vii Ct vs Well Position view 63 Ct See threshold cycle curves amplification 54 dissociation 64 77 79 melting 64 standard 10 75 Custom TaqMan Gene Expression Assays 14 customer feedback on Applied Biosystems documents viii D DANGER description xi data Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 91 Index analyzing 60 exporting 70 generating PCR data from AQ plates 31 45 importing 25 39 omitting froma plate 66 deltaRn 4 Delta Rn vs Cycle view 63 derivatives 78 designing AQ experiments determining reagent configuration 11 primers and probes 14 selecting an SDS chemistry 11 Detector Manager dialog box 73 detector task 38 detectors adding to AQ plates 26 40 creating 73 definition 38 73 deviation standard 56 display options 60 dissociation curves 64 77 79 documentation related viii documents AQ Plate 24 38 exporting 70 templates 25 39 dyes passive reference 27 41 quencher 62 reporter 4 62 ROX 27 41 62 SYBR Green 11 14 TAMRA 62 74 E endpoint PCR 2 equipment 5 example AQ experiment AQ Plate document example 28 42 chemistry 14 overview 7 PCR master mix 23 37 procedure 82 reagent configuration 14 reverse transcription 18 Expert Mode 43 45 exporting AQ plate data 70 F Fast system
98. to 100 ng Equal to 9 0 Equal to 45 0 Equal to 333 0 Nuclease free water Total 20 0 100 0 740 0 One master mix was prepared for each of the six standards 4 replicates plus extra volume for pipetting losses One master mix was prepared for each of the two populations being studied 86 samples plus extra volume for pipetting losses Unknowns target sequences being quantitated and standards were arranged on a plate For 7500 Fast system 20 uL of the appropriate PCR master mix containing cDNA was added to each well The plate was kept on ice until it was loaded in the 7500 Fast system RNase P Population 1 NTC STD 1250 STD 2500 STD 5000 STD 10000 STD 20000 RNase P Population 2 GR2478 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 37 Chapter 5 Running an AQ Plate 7500 Fast System Creating an AQ Plate Document Creating an AQ Plate Document Overview An AQ plate document stores data collected from an AQ run for a single plate AQ plate documents also store other information about the run including sample names and detectors Run Setup For each AQ plate document that you create specify detectors standards and detector Requirements tasks e A detector is a virtual representation of a gene specific nucleic acid probe reagent used in assays You specify which detector to use for each target seq
99. tting Started Guide for 7300 7500 7500 Fast Systems 93 Index wells replicate 10 width lines 60 workflow AQ experiment overview 3 example AQ experiment 82 X x axis 60 Y y axis 60 94 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 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 site at www appliedbiosystems com Applera is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses 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 07 2006 Applied Biosystems Part Number 4347825 Rev E
100. tup refrigerate or freeze the reaction plate until you can load and run it on the 7500 Fast instrument Low AR or R values Extension time is too short Use the default thermal profile settings See page 44 Primer dimer formation To ensure optimal results run the reaction plate as soon as possible after completing the reaction setup If you cannot run a reaction plate within 2 hours after completing the reaction setup refrigerate or freeze the reaction plate until you can load and run it on the 7500 Fast instrument 48 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 5 Running an AQ Plate 7500 Fast System Troubleshooting Troubleshooting Observation Possible Cause Action Run takes more than 40 minutes Thermal cycler mode is set to Make sure that the thermal cycler Standard or 9600 Emulation mode is set to Fast see page 44 Rn vs Cycle plot is not displayed ROX dye was not selected as the Select ROX dye as the passive passive reference when the plate reference when you set up the plate document was set up document Extremely high AR or R values ROX dye was not selected as the Select ROX dye as the passive passive reference when the plate reference when you set up the plate document was set up document Evaporation Make sure that the reaction plate is sealed completely especially around the edges High var
101. uence Appendix A on page 73 explains how to create detectors e A standard is a known amount of a target sequence You must have a set of standards for each target sequence on the plate e A detector task specifies how the software uses the data collected from the well during analysis You apply one of three tasks to each detector in each well of a plate document Task Symbol Apply to detectors of Unknown m Wells that contain target sequences that you are quantitating Standard E Wells that contain samples of known quantities NTC M Negative control wells that contain PCR reagents but that lack template 38 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Creating an AQ Plate Document You can enter sample information into a new plate document copy and paste sample information from existing plate documents import sample information from existing plate documents or use a template document to set up new plate documents This section describes setting up new plate documents Refer to the Online Help for information about copying or importing sample information from existing plate documents or using template documents Note The following procedure is illustrated using the example experiment data file see page 7 To create a new AQ plate document 1 Select Start gt All Programs gt Applied Biosystems 7500 Fast System gt 7500 Fast w
102. uide for 7300 7500 7500 Fast Systems 67 NX Chapter 6 Analyzing AQ Data Omitting Samples FEH 68 Click or select Analysis gt Analyze to reanalyze the run without the outlier data Repeat steps 4 and 5 for other wells you want to screen Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Sek p Y Inetiument Y Results Fla Y Spela YC imipu enl f Ampliar Pha 5 S and rd Cune Y Dis u ali u Y Rep il Ct va Woell nziti n dz J 40 C Outlier removed 25 E EE ee ee m L T Hw a A g iS eg eee mig 0 l 4 7 10131619 222528 3 34 37 41043 16 1950 5555616 BY TOTS TBO es 85 8891 oF Wel P siti r Cjo T F Chapter 6 Analyzing AQ Data E Omitting Samples PE Removing Outliers on the Standard Curve 1 Select the Standard Curve tab 2 Select all wells in the plate grid 3 Examine the curve for outliers Note To zoom in click Zoom In then click the standard curve plot or click drag to zoom in on a group of wells 4 Ifan outlier is present click Select then click the outlier on the standard curve plot to highlight the well or click drag to highlight a group of wells 5 Click Omit Wells or right click then select Omit Wells 6 Click gt or select Analysis gt Analyze to reanalyze the run without the outlier data T Repeat steps 4 through 6 for other o
103. utliers you want to remove Setup Instrument Results Plate Y Standard Curve Dissociation Y Report Standard Curve Amplification Plot 30 000 29 500 29 000 28 500 28 000 Ton Slope 3 577242 ntercjept 41 033016 R2 1 997959 Highlighted Wells Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems 69 eee l Chapter 6 Analyzing AQ Data PRE Exporting AQ Plate Data Exporting AQ Plate Data You can export numeric data from AQ plates into text files which can then be imported into spreadsheet applications such as Microsoft Excel software You can export graphs as a Microsoft PowerPoint software presentation or as JPEG files Note You must have PowerPoint installed for the export graphs to PowerPoint feature to work To export data to a spreadsheet application 1 Select File gt Export then select the data type to BEA view Tools Instrument Analysis Window Hep export New CrN R Open Chrl o e Sample Setup txt Close e Calibration Data csv Save AS Import Sample Setup e Spectra csv Sample Setup e Component csv Page Setup ae Print Preview Spectra e Delta Rn csv Print Ctrl F Component Delta Rn Ct csv 1 RNAse a Exit Dissociation e Dissociation csv e Results Results R
104. ye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves b Prepare the cDNA plate by pipetting into each well of the plate e 10 uL RT master mix e 10 uL RNA sample Convert up to 2 ug of total RNA to cDNA per 20 uL reaction RT Master Mix Standard Plate Component uL Reaction ceili 10X Reverse 2 0 54 Transcription Buffer 25X dNTPs 0 8 21 6 10X random primers 2 0 54 MultiScribe Reverse 1 0 27 Transcriptase 50 U uL Nuclease free water 4 2 113 4 Total 10 270 Each RT reaction is 20 uL see step 3b If you need 5 uL of cDNA at 50 uL total volume for each of 104 PCR reactions per plate see step 4 you need 27 RT reactions Extra volume is included to account for pipetting losses as well as extra cDNA for archiving O O 00000666 00000668 00000068 00000066 00000066 00000066 000000686 000000686 00000066 000000686 000000686 82 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems c Program the thermal cycler using the indicated parameter values for the RT step of the two step RT PCR method Appendix E Example AQ Experiment Note You have the option to use one step RT PCR as explained in Selecting One or Two Step RT PCR on page 12 RT reactions are performed with Standard Universal Master Mix f
105. ying Thermal Cycling Conditions and Starting the Run 3 Select File gt Save enter a name for the AQ plate document then click Save Optional If you want to use this plate document again save it as a template document Select File gt Save As In the Save in drop down list navigate to Applied Biosystems 7300 7500 7500 Fast System Templates Type the File name then select sdt for Save as type to save the file as a template 4 Load the plate into the instrument Note The A1 position is in the top left side of the instrument tray 5 Click Start As the instrument performs the PCR run it displays real time status information in the Instrument tab and records the fluorescence os 7300 7500 system emissions Notched top right corner for standard After the run the status values and buttons are plates grayed out the Analysis button is enabled and a message indicates whether or not the run is successful All data generated during the run are saved to the AQ plate document that you specified in step 3 7500 Fast system Notched top left corner for Fast plates 32 Absolute Quantitation Using Standard Curve Getting Started Guide for 7300 7500 7500 Fast Systems Chapter 5 Running an AQ Plate 7500 Fast System Before You Begin See page 34 Introduction Prepare the See page 34 PCR Master Mix Designing an AQ Experiment Prepa

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