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Relative Quantification Getting Started Guide for the Applied

1. 2 e0e00000000 80 1 1 ee ee00e0000600080 a Label the reaction plates ensuring that you 000000000000 include an endogenous control on each CD CD ED ples EEEE XEJEEE plate 000000000000 GISISTO SISISIS SIOTOTO H E Gogenous SeeeeeeeOood b Pipette 50 uL 20 uL for the Fast system of controls GAPDH Fast system plates are the appropriate PCR master mix containing the same except the notched corner is at eee0e00800000080 cDNA into each well of the plate Sana A GEE the n left i n an 2 ee 000000080 position Instead o e c Keep the reaction plates on ice until you are SSIS OOTO STTS ole top right and Fast ready to load them into the 7300 7500 7500 i R i A 5 n plates have a 0200000 00000 recommended 30 uL Fast system 00000008 ae N volume capacity e ee000000080 2ee0e0080008000080 CIVISIS G1SISIS CISIST cc C1VIGIS SISISTS GIVI pies e e e0e2000006808 eee0e0000600080 ee ee00080006080 00000000 a S 6 Create an RQ Plate document as described in New Document Wizard Creating a Relative Quantification RQ Plate felne Ducesient Document on page 4 1 a container and template for the document and enter the operator name and SS T E Assay Relative Quantification ddCt Plate IMPORTANT All plates added to a study g Container J6 w ell Clear must have identical thermal cycling a i
2. Creating an RQ Study Document To conduct a comparative analysis of RQ plates in a study or to analyze a single RQ plate experiment you must first create an RQ Study document IMPORTANT RQ Study software is an optional package for the 7300 instrument but is standard for the 7500 and 7500 Fast instruments IMPORTANT RQ plates run with standard thermal cycling conditions Fast thermal cycling conditions cannot be combined into a single RQ study The SDS software uses the comparative Ct method 2 4 of relative quantification For more information about methods of calculating relative quantification refer to ABI PrisM 7700 Sequence Detection System User Bulletin 2 PN 4303859 In an RQ study you can You cannot e Select the endogenous control and the calibrator sample Create add or modify samples e Select the control type when applicable e Create add or modify detectors e Set baseline and threshold values and RQ Min Max e Change detector tasks Confidence Levels You can perform these operations in RQ Plate e Omit individual wells or sample replicates documents To create a new RQ Study document 1 m Select File gt New New Document Wizard Define Document 1 Select the assay container and template for the document and enter the operator name and 2 Inthe Assay drop down list of the New ce Document Wizard select Relative j Assay Relative Q
3. File Add To Plate Document Hew Duplicate a Add to Plate Document Import Export Clear Clear All Properties Hew Detector Name e Description 4 z a Quencher Dye inon o Color Hi o Notes Create Another Cancel Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 81 Appendix A 5 10 11 g 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 quencher for TaqMan probes and None for TaqMan MGB probes Click the Color box and select a color to represent the detector using the Color dialog box Optionally click the Notes field then enter any additional comments for the detector Click Create Another if you want to create another detector Click OK to save the detector and return to the Detector Manager Repeat steps 2 through 9 for the remaining detectors In the Detector Manager click Done when you finish adding detectors Example Experiment In the example RQ experim
4. Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 63 Chapter 6 Analyzing Data in an RQ Study Configuring Analysis Settings c RQ Results panel Contains the three results based tabs Plate default Amplification Plot and Gene Expression Note You can save the RQ Study document now or wait until after specifying analysis settings and analyzing the data Configuring Analysis Settings After you create the RQ Study document 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 explained below 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 and threshold as explained in Manual Baseline and Threshold Determination on page 66 To configure analysis settings Click m or select Analysis gt Analysis Settings 2 Inthe Detector drop down list select AIl 3 Select Auto Ct The SDS software automatically generates baseline and threshold values for each well IMPORTANT After analysis you must verify that the baseline and threshold were called correctly for each well as explained in Adjusting the Baseline a
5. Task Symbol Apply to detectors of Target Wells that contain PCR reagents for the amplification of target sequences Endogenous E Wells that contain reagents for the amplification of the Control endogenous control sequence Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 31 Creating an RQ Plate Document You can enter sample information into a new plate document 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 importing sample information or using template documents To create a new plate document 1 Select Start gt Programs gt Applied Biosystems 7300 7500 gt Applied Biosystems 7300 7500 SDS Software Ee to start the SDS software 2 Select File gt New 3 In the Assay drop down list of the New Document Wizard select Relative Quantification ddCt Plate 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 You cannot use RQ Plate documents to perform relative quantification using the relative standard curve method The information stored in AQ and RQ Plate documents is not interchangeable 4 Enter a name in the Default Plate Name field o
6. U An air bubble lies at the bottom of the well because the plate was not centrifuged with sufficient force or for sufficient time Place the reaction plates on ice until you are ready to load them into the 7500 Fast system Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 45 Example Experiment Primers and probes for the example RQ experiment are obtained from the TaqMan Gene Expression Assays product line and are provided as a 20X Gene Expression Assay Mix The PCR master mix is prepared as follows Chapter 5 Generating Data from RQ Plates 7500 Fast System N LN CHEMICAL HAZARD TaqMan Universal PCR Master Mix 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 to account for pipetting losses CDNA are added to each well o ee0e00000060 80 o ee000006000 80 000000000000 e0e000080806000 samples o ee0e000060000 80 ee0e0e000e0000 80 GOO 0 OOOO OOO 8 Endogenous XXXZZ JS controls GAPDH EEKE EKEKEEE EEEF EKEJ EREE 000000000000 EEKE EKXEKEEE samples EEK XEXEKEEEE ee0e000e00006080 0 000000000000 Endogenous SIOIOI GISISTO O O OF controts GAPDH a Contains forward and r
7. Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Generating Data from RQ Plates 7300 or Standard 7500 System Chapter 4 Workflow Prepare the See page 28 PCR master mix Prepare the See page 29 reaction plate Create a new See page 31 RQ Plate document Reverse Program the See page 35 Scrip thermal cycling conditions RQ Plate document Generating data from Generating data from D RQ Plates iit ST RQ Plates Standard Fast See page 37 Start the run View RQ plate data See page 38 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 27 ae Chapter 4 Generating Data from RQ Plates 7300 or Standard 7500 System ANDA RAID Before You Begin Before You Begin Check that background and pure dye runs have been performed regularly to ensure optimal performance of the 7300 7500 system For more information about calibrating the 7300 7500 system refer to the Online Help located within the software by clicking the 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 The TaqMan Universal PCR Master Mix Protocol PN 4351891 explains how to use the reagents in the kit The following table l
8. WARNING description vii Well Information tab 72 wells replicate 17 width bars 74 width lines 38 56 73 74 workflow RQ experiment overview 3 X x axis 38 56 73 74 Y y axis 38 56 73 74 90 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Science iScience To better understand the complex interaction of biological systems life scientists are developing revolutionary approaches to discovery that unite technology informatics and traditional laboratory research In partnership with our customers Applied Biosystems provides the innovative products services and knowledge resources that make this new Integrated Science possible 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 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 www appliedbiosystems com Applied Bibsystems 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 busines
9. background in amplification curve 66 bar width 74 baseline adjusting 66 definition 3 examples 67 C calibrating the 7300 7500 instrument 28 42 calibrator definition 17 selecting in RQ studies 65 CAUTION description vii cDNA generating 25 storing 26 See also reverse transcription Chemistries 19 comparative method of calculation 3 62 Index Component view 39 57 concentration of RNA 24 confidence levels 65 conventions text vii Ct See threshold cycle curves amplification 66 standard 2 D data analyzing 38 56 72 exporting 40 58 79 generating PCR data from RQ plates 36 54 importing 32 48 omitting from a study 77 delta Rn 4 Delta Rn vs Cycle view 73 designing RQ experiments determining reagent configuration 19 PCR method 16 primers and probes 21 selecting the chemistry 19 Detector Manager dialog box 81 detector tasks definition 31 47 detectors adding to RQ plates 32 49 creating 81 definition 81 selecting for RQ studies 64 deviation standard 68 display options 38 56 73 74 documentation feedback 1x documents exporting 40 58 79 RQ Plate 31 47 RQ Study 62 templates 32 48 dyes FAM 21 82 reporter 4 ROX 33 39 50 57 SYBRGreenI 19 21 TAMRA 39 57 82 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 87 Index E emulation mode 9600 37 endogenous controls associating with detectors 31 47 definit
10. Relative Quantification Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Introduction and Example RQ Experiment Designing an RQ Experiment Performing Reverse Transcription Generating Data from RQ Plates Standard Generating Data from RQ Plates Fast System Generating Data in an RQ Study Copyright 2004 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Authorized Thermal Cycler This instrument is an Authorized Thermal Cycler Its purchase price includes the up front fee component of a license under United States Patent Nos 4 683 195 4 683 202 and 4 965 188 owned by Roche Molecular Systems Inc and under corresponding claims in patents outside the United States owned by F Hoffmann La Roche Ltd covering the Polymerase Chain Reaction PCR process to practice the PCR process for internal research and development using this instrument The running royalty component of that license may be purchased from Applied Biosystems or obtained by purchasing Authorized Reagents This instrument is also an Authorized Thermal Cycler for use with applications licenses available from Applied Biosystems Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents Purchase of this product does not itself convey to the purchaser a complete license or right to perform the PCR process Further
11. 0 021 0 077 Cycle Number Notes 68 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Adjusting the Baseline and Threshold Manual Baseline and Threshold Determination a iad 2lo E E Omit Data Delta Rn ace E Calibrator liver Orientation Det a ae Plot Gene Expression ES Detector Threshold Auto ct Baseline Auto ACRI 0 535276 ACYRZ O353770 Auto Auto 0 056001 Auto 0 267214 Delta Rn vs Cycle 1 0e 001 exponential phase of the 1 0e 000 0 200000 amplification FLT4 0 451420 Auto Auto 1 0e 001 curve GAPDH 0 418936 Auto Auto GTF28 0 254278 Auto Auto GTFal 0 280024 Auto Auto rm ee 10e002 HTR4 0 082686 Auto Auto z 1 0e 003 Sample Detector Task 1 0e 004 Liver COSD Target 27 437 6439 01271 0 000 1 000 Jo3 5 fF 8 1713 75 78 19 21 23 25 2F 29 31 33 35 SF 39 Kidney CD3D Target 28 158 8190 0 194 2 051 0 241 Bladder CD3SD Target 31 519 10 007 0 172 Cvrcle Mumher ap Analpes Settings Auto Ct f Manual Ct E Threshold 2b 214 fs Start cycle Auta End cycle Auta Y Sample Summary Well Information j 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 Inthe RQ Detector grid select a detector The SDS software displa
12. Choose probe and primer sets for both your target and endogenous control sequences Applied Biosystems provides three options for choosing primers and probes TaqMan Gene Expression Assays Provide you with optimized ready to use TaqMan reagent based 5 nuclease assays for human mouse or rat transcripts For information on available primer probe sets go to http www allgenes com TaqMan Custom Gene Expression Assays Designs synthesizes formulates and delivers quality controlled primer and probe sets Use this service if the assay you need is not currently available To place an order contact your Applied Biosystems representative or go to http www allgenes com Primer Express Software Helps you design primers and probes for your own quantification assays For more information about using this software refer to the Primer Express Software User s Manual Applied Biosystems provides assay design guidelines that have been developed specifically for quantification assays When followed these guidelines provide a reliable system for assay design and optimization For information about the assay design guidelines refer to the SDS Chemistry Guide If you ordered TaqMan Gene Expression Assays or TagMan Custom Gene Expression Assays probes are already labeled with FAM as a reporter dye If you design your own assays you need to specify a reporter dye for your custom probe s For singleplex experim
13. Cleavage Polymerization Completed e Optimized assays available Q Q gt a J e Allows 5 nuclease assay to be carried out 5 iv 5 3 M 3 5 during PCR 2 p J 5 5 Step 2 During each extension Step 3 After being separated from cycle the AmpliTagq Gold DNA the quencher the reporter dye polymerase cleaves the reporter emits its characteristic dye from the probe fluorescence SYBR Green l reagents T T T T Step 1 Reaction setup ani A A E D A Description The SYBR Green dye fluoresces when bound to Uses SYBR Green dye a double stranded DNA double stranded DNA binding dye to detect PCR products as they a E AN accumulate during PCR cycles A 9 y O When the DNA is denatured Advantages the SYBR Green dye is released and the fluorescence e Reduces cost no probe needed is drastically reduced e Amplifies all double stranded DNA E PRIMER r Step 3 Polymerization e Yields a melting profile of distinct PCR runs During extension primers e Increases signal fluorescence as amplification e eas ies proguet product length increases naaa j Limitations i PE ss i oe r Binds nonspecifically to all double stranded DNA o 2 o SS a e a sequences To avoid false positive signals check D a aa oe oe a pene Dee ene f ifi ducks ti naa iati tl inna e E A a double stranded product or nonspecific product formation using dissociation resulting in a net increase in
14. Experiment Chapter 1 Overview Introduction and Example RQ Experiment About the 7300 7500 7500 Fast See page 2 system About relative quantification See page 2 About RQ experiments See page 3 Example RQ Experiment See page 6 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 1 Chapter 1 Introduction and Example RQ Experiment About the 7300 7500 7500 Fast System About the 7300 7500 7500 Fast System Description Relative Quantification 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 quantification in fewer than 40 minutes The 7300 7500 7500 Fast system allows the user to perform several assay types using plates or tubes in the 96 well format This guide describes the relative quantification RQ assay type For information about the other assay types refer to the Sequence Detection Sy
15. Run Mode Standard 7500 z Stage 3 Step 2 60 0 1 00 Note Refer to the Online Help for instructions on Add cycle Add Hold Add Step modifying thermal cycling parameters E tl PP Data Collection Times and Temperatures One step RT PCR Initial Steps PCR Each of 40 Cycles Reverse Transcription AmpliTaq 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 To specify thermal cycling conditions and start the run 1 Select the Instrument tab Instrument Control Temperature By default the standard PCR conditions for the ay G a ise icy freee sich ee PCR step of the two step RT PCR method are mE TE ER di spl ay e d Time ma 2 Step Thermal Cycler Protocol 2 Verify that Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 Stage 3 e For two step RT PCR the default PCR thermal cycling conditions are set e For one step RT PCR you set the thermal cycling parameters as shown above e Sample volume is 50 uL Add Cycle Add Hold Add Step Add Dissociation Stage Settings Sample Volume pL 20 Run Mode Standard 7500 X Stage 3 Step 2 60 0 1 00 bd Help Data Collection Notes 36 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Specifying Thermal Cycling Conditions and Starting the Run Th
16. Target Wells that contain PCR reagents for the amplification of target sequences Endogenous g Wells that contain reagents for the amplification of the Control endogenous control sequence Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 47 Chapter 5 Generating Data from RQ Plates 7500 Fast System Creating a Relative Quantification RQ Plate Document Creating an RQ Plate Document You can enter sample information into a new plate document 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 importing sample information or using template documents To create a new plate document 1 Select Start gt Programs gt Applied Biosystems RRR 7300 7500 7500 Fast gt Applied Biosystems Delne Document 7300 7500 7500 Fast SDS Software i to a container and template for the document and enter the operator name and start the SDS software 2 Select File gt New Container 96 ell Clear Y Template Blank Document ae 3 Inthe Assay drop down list of the New o Document Wizard select Relative Operator Administrator Quantification ddCt Plate Accept the default Comments settings for Container and Template 96 Well Clear and Blank Document J Pes IMPORTANT You c
17. 7 00 Liver T 514 Liver 7 83 Liver H 2 46 Liver T 6 38 Liver 6 85 Liver TERN Liver rs Liver M259 Liver Tsis Liver Hes Liver H i Setup Instrument Y Results Plate Spectra Y Component Amplification Plot Component Sa BEE eo ee ee 3200 00 2800 00 2400 00 2000 00 1600 00 Fluorescence 1200 00 800 00 400 00 Cycles Component s Mv Fam WY Rox Setup Instrument Results Plate Spectra Component Amplification Plot Rn vs Cycle Sasa Ua base HES Feet Hace M denM Bea A Pepe am O Ceara Cycle Number PAS Foie N BEREC IC estos fan a Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Detector Al z Line Color well Color z 57 Chapter 5 Generating Data from RQ Plates 7500 Fast System Exporting RQ Plate Data Reanalyzing Data Raw fluorescence data spectra R values and well information sample name detector and detector task are saved in an RQ plate document If you decide to omit wells or change well information after a run is complete you must reanalyze the data Note After the software analyzes data the Analyze button is disabled Whenever you change a setting that requires reanalysis the Analyze button is enabled Exporting RQ Plate Data You can export numeric data from RQ
18. 96 Well Spectral Calibration Kit 1 PN 4360788 7500 Fast System 96 Well Spectral Calibration Kit 2 PN 4362201 Optical 96 Well Fast Thermal Cycling Plate with Barcode code 128 PN 4346906 TaqMan RNase P Fast 96 Well Instrument Verification Plate PN 4351979 TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG PN 4352042 TaqMan Gene Expression Assays PN 4331182 IMPORTANT RQ plates run with standard thermal cycling conditions Fast thermal cycling conditions cannot be combined into a single RQ study 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 reagents Users of the 7300 7500 system must use standard TaqMan Universal PCR Master Mix 2x for an approximately 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 use of the TaqMan Fast Universal PCR Master Mix 2x allows 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 PN4351891 IMPORTANT If you are using TaqMan Fast Universal PCR Master Mix you must start the run within 4 hours of preparing the plate ANIN CHEMICAL HAZARD TaqMan Fast Universal PCR Master Mix may cause eye and skin irritation Exposure may cause discomfort if
19. Over Cker hier Lee Uver Lee Uver Lee Uver LR Liver E Uvar Ler Uvar kli Ler Uver Lier Uver Lier Uvar Ler E Fluver Lee Over L Lke Uver Lee Uver Lee Uver Lee Uver Lie al a ue Lee Over Lwer Uver Lee Uver Lee Uver Lee Uver Lre H uver ower Over Lre Uver Lee Uver Lee Uver Lee Uver Lre H E B m E i E E a E 11 Example RQ Experiment 8 Start the RQ run a Select the Instrument tab By default the Fast PCR conditions for the PCR step of the two step RT PCR method are displayed for the 7500 Fast instrument whereas the Chapter 1 Introduction and Example RQ Experiment 23 7500 Fast System SDS Software Plate1 Relative Quantification Plate D File View Tools Instrument Analysis Window Help Nel 4a 82s Instrument Control star Estimated Time Remaining hh mm R Temperature Hest Sink Block Sample Cover standard PCR conditions are displayed for a Ae ae F the 7300 and 7500 standard instruments not ees i shown Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage1 Stage 2 Reps 1__ Reps 95 0 _ Add Cycle Add Hold Add Step Help Settings 20 Run Mode Fast 7500 v Stage 2 Step 2 60 0 0 30 x Sample Volume pL Data Collection b Select File gt Save As enter a name for the RQ Plate document then click Save c Load the plate into the instrument d Cli
20. Plate Document c Click Next gt T Specify the detectors and tasks for each well cance x Ectup Same Pate a Click a well or group of wells for Setup che sample pace ath tases quant ie and detectors Lee Detector Reparar Guencher loak Color Tara Tab tn n Tater W ACR FA none Target z BL en one B Target tr Saget ENDO 4 s r 2 replicates to select it b Click to select the detector s for the well Aoo mip c Click under the Task column to assign the detector task d Select Use e Click Finish EE il bl ba cl al Ka fan car You cannot add RQ plates to RQ studies unless you have specified sample names as indicated in the message shown to the right You can enter sample names by using the Well Inspector or by using the Click OK in place sample name editing Feature select wellis and type text directly into the sample name SDS v1 3 Note You will need to enter sample names before saving the RO Plate document After editing sample name s you can then save the RO Plate document The SDS software creates the plate document and displays the Well Inspector 8 Enter the sample names a In the Well Inspector click a well or click drag to select replicate wells Ce m Liver Liver b Enter the sample name c Ifnecessary change the setting for the Passive Reference dye By default the R
21. Plot or Gene Expression analysis results are displayed E od 2 2 c 3 G o T EA S 1 E o o D 3 al a lt a z 3 Sample Summary Well Information Detectors vR1 VR2 CR2 cD3D FLT4 GAPDH To see information about a specific well select the Well Information tab Example Experiment Suppose that you want to view the comparative gene expression levels of the following genes when the liver tissue is used as the calibrator ACVR1 ACVR2 CCR2 CD3D and FLT4 Selecting the detectors in the RQ Detector grid 1 displays the sample information in the RQ Sample grid 2 and in a result graph in the RQ Results panel 3 Note that e The Gene Expression tab is selected and the gene expression levels are sorted by detector e Gene expression levels for bladder samples are indicated by the green bar those for kidney samples by the blue bar These colors also indicate the samples in the RQ Sample Grid and the RQ Results Panel plots e Because liver samples are used as calibrators the expression levels are set to 1 But because the gene expression levels were plotted as log values and the log of 1 is 0 the expression level of the calibrator samples appear as 0 in the graph e Because the relative quantities of the targets are normalized against the relative quantities of the endogenous control the expression level of the endogenous control is 0 there are no bars for GAPDH
22. Thermal Profile Auto Increment Ramp Rate e For two step RT PCR the default PCR Stage1 stage 2 Reps 1__ Reps 40 thermal cycling conditions are set e Sample volume is 20 uL Add Cycle Add Hold Add Step Help Settings Sample Volume UL 20 Run Mode Fast 7500 Data Collection Stage 2 Step 2 60 0 0 30 v Settings Sample olume pL 20 Run Mode Fast 500 Fast 7500 Standard 7500 9600 E mulation Data Collection Stage 2 Step 2 60 0 0 30 e Fast 7500 is selected as the run mode Note If you are using SYBR Green I reagent chemistry and you want to determine if there is contamination or if you want to determine the dissociation temperature create a separate Dissociation assay or template Refer to the Online Help for more information Users of the 7500 Fast System can use SYBR Green I reagents with Standard or 9600 Emulation run modes 3 Select File gt Save As enter a name for the RQ Plate document then click Save Notes 54 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Specifying Thermal Cycling Conditions and Starting the Run 4 Load the plate into the precision plate holder in the instrument Ensure that the plate is properly aligned in the holder Note The A1 position is in the top left of the instrument tray The bar code is t
23. Universal PCR Master Mix 2x 25 0 125 0 1X 20X Gene Expression Assay Mix 2 5 12 5 1X cDNA sample 5 0 25 0 50 ng for the 50 uL reaction Nuclease free water 17 5 87 5 Total 50 0 250 eeeeeee e eeeceeceo e ee eeeeeo Seeeeeee e ee eeeee eee eeeeceo eeeeece eeeeeeee Oe eee6ee Ooeeee6ce oeeee6cee OSeeeeeee e eeeeee e ee eeeeceo e eeeeeceo Seeeeeee eee eeeeeo eeeeoeeceo eee eececeo Seeeeeeee Oe eeeee oeeeeee oeeee6e OLIA Liver samples Endogenous controls GAPDH Kidney samples Endogenous controls GAPDH a Contains forward and reverse primers and labeled probe b 24 master mixes are prepared one for each of 23 genes plus the endogenous control Volume for five reactions 4 replicates plus extra to account for pipetting losses Samples and endogenous controls are arranged on three plates as shown below 50 uL of PCR master mix containing cDNA are added to each well Bladder samples 0000 000868000 0000 00008608 0000 80088080080 0000 00800808 0000 0008000080 0000 0008080880 0000 008080008 TAEAE IAS Endogenous The reactions are kept on ice until the plates are loaded on the 7300 7500 7500 Fast system controls GAPDH Notes 30 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Creating a Rel
24. Viewing RQ Plate Data Starting the Analysis To analyze RQ Plate data after the run click or select Analysis gt Analyze The SDS software mathematically transforms the raw fluorescence data to establish a comparative relationship between the spectral changes in the passive reference dye and those of the reporter dyes Based on that comparison the software generates four result views Plate Spectra Component and Amplification Plot About the Results Tab In the Results tab you can view the results of the run and change the parameters For example you can omit samples or manually set the baseline and threshold If you change any parameters you should reanalyze the data The Results tab has four secondary tabs each of which is described below Details are provided in the Online Help e To move between views click a tab e To select all 96 wells on a plate click the upper left corner of the plate e To adjust graph settings double click the y or x axes of a plot to display the Graph Settings dialog The adjustable settings depend on which plot you are viewing Notes F Fie view Tools Instrument Analysis Window Help Bp lA Blo E setup Y instrument Yresuts Y 7 Plate Spectra Y Amplification Plot B Liver Liver Liver Liver ia Liver 5 87 5 95 5 95 5 97 6 52 6 265 Graph Settings Real Time Settings Post Run Settings Y Axis Auto Scale Minimums Y Axis V Auto Scale C Linear Minimum 0
25. You can adjust graph settings for gene expression plots in the Graph Settings dialog box including e Bar width e 3D bars e Autoscaling e Data display as Log RQ or Raw RQ To access the Graph Settings double click on one of the axes Refer to the Online Help for more information about adjusting graph settings for gene expression plots Gene Expression Plot Orientation Detector Detectors are plotted on the x axis and each bar shows the detector value of a single sample Notes fe Omit Data Deta Rn vs Cycle Calibrator Liver Orientation Graph Settings Bar Width zo 0 100 Group Spacing 70 0 100 Group Spacing only applicable if Bar Width is lt 100 30 Bars SampleDetector Name In Bar W Auto Scale Minimum Masimum sw cea Y Ass Data Logi AO C Raw AG Defaults Detector 7 Plate Amplification Plot Y Gene Expression Log 10 Relative Quantification 0 pp amp g CCR2 CD3D FLT4 g r GAPDH Detectors 14 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Analyzing and Viewing the Results of the RQ Study Error Bars for Gene Expression Plots Gene Expression Plot Orientation Sample E Omit Date Dera An ve Cycle gt Calibrator Liver gt Orientation 7 Plate Amplification Plot Y Gene Expression Samples are plot
26. data 2 Kidney types are exported for existing plates Exit 2 Enter a file name for the export file Note The name of the dialog box depends on the type of data you want to export 3 Click Save Notes 40 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Generating Data from RQ Plates Chapter 5 7500 Fast System Workflow Prepare the See page 42 PCR master mix Prepare the See page 44 reaction plate Create a new See page 47 RQ Plate document Specify the See page 52 thermal cycling conditions Save the See page 54 RQ Plate document Generating lt gt Generating data from data from RQ Plates eee RQ Plates Standard Fast Start the run See page 55 View RQ plate data See page 56 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 41 Chapter 5 Generating Data from RQ Plates 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 Fast System Requirements e 7500 Fast System with fast hardware and software Sequence Detection Systems Software v1 3 e Fast reagents and plastics 7500 Fast System
27. experiment is to compare the levels of expression of 23 genes in the liver kidney and bladder tissue of an individual The experiment is designed for singleplex PCR Samples and endogenous controls were amplified in separate wells Glyceraldehyde 3 phosphate dehydrogenase GAPDH serves as the endogenous control Four replicates of each sample and endogenous control are amplified In this experiment an entire 96 well plate is devoted to each tissue because the four replicates of each of the 23 genes plus the endogenous control require all 96 wells Predesigned and labeled primer probe sets are selected from the Applied Biosystems TaqMan Gene Expression Assays product line Reactions are set up for two step RT PCR where the High Capacity cDNA Archive Kit and the TaqMan Universal PCR Master Mix are used for reverse transcription and PCR respectively Data are generated by running three RQ plates one for each tissue All three plates are analyzed in an RQ study with the liver samples serving as the calibrator Notes 6 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Example RQ Experiment Procedure 1 Design the experiment as explained in Chapter 2 on page 15 a Designate the targets calibrator endogenous control and replicates b Order the reagents for TaqMan probe based chemistry c Order the appropriate TaqMan Gene Expression Assays
28. for details f fy Start cycle Auto End cycle Auto Gene Expression If you know the optimal baseline and Caibetor Sarre EE threshold settings for your experiment you Do ales En can use the Manual Ct and Manual Baseline Control Type 7 ues options RO Min Max Confidence 35 00 Replicate and Outlier Removal W Remove Outlier OK amp Reanalyze Cancel Apply b If necessary manually adjust the baseline and threshold Note See Adjusting the Baseline and Threshold on page 66 c Click or select Analysis gt Analyze to reanalyze the data Baseline is set before Threshold falls within amplification begins exponential phase of curve d View analysis results by clicking a tab in the 5 SR BE By omit Dete Der Anvs yde s Calbretor iver Orientation Detecior RQ Results pane e If desired save the RQ Study document Conclusion As shown in the figure on the right expression levels of CCR2 are greater in the liver than in the kidney or bladder tissues of this individual Cc Ss 2 rz w E 3 Ga a sis ac o oe oO faz t ai E v lt gt Sample Summary Well Information Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 13 Chapter 1 Introduction and Example RQ Experiment Example RQ Experiment Notes 14 Relative Quanti
29. function properly and may be crushed when using the 96 Well Fast Block Fast Plates Standard Plates Standard plate has S notch at top right SSS corner by A12 plate has notch at top left corner by A1 FTT UVV VV VVU Vaan reaction reaction volume volume To Prepare the Reaction Plate 1 Label the reaction plates ensuring that you include an endogenous control for each sample type for example each tissue in a study comparing multiple tissues If samples are spread across multiple plates each plate must have an endogenous control Additionally every plate must include an endogenous control for every sample type on the plate 2 Into each well of the reaction plate add 20 uL into each well of the low head space reaction plate of the appropriate PCR master mix 3 Seal the reaction plate with an optical adhesive cover e ABI PRISM Optical Adhesive Cover quantity 100 PN 4311971 e Optical Adhesive Cover quantity 25 PN 4360954 4 Centrifuge the plate briefly 5 Verify that each reaction is positioned in the bottom of the well Notes 44 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Notes Preparing the Reaction Plate Correct Position Incorrect Positions GR1303 The reaction is positioned correctly in the bottom of the well U The reaction lies on the side wall because the plate was not centrifuged
30. investigators and laboratory staff who conduct Guide relative quantification studies for gene expression using the Applied Biosystems 7300 7500 7500 Fast Real Time PCR 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 If you want to integrate the 7300 7500 7500 Fast system into your existing laboratory data flow system you need networking experience Text Conventions Bold indicates user action 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 gt Spot Set User Attention The following user attention words appear in Applied Biosystems user documentation Words Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a c
31. 00 1 000 0 828 Kidney CD3D Target 28 0207 2 008 0 249 0 002 0175 Bladder CD30 Target 31 585 10 216 0193 3847 0o70 0 002 0 050 0 096 0 135 7 441921617 1974 939507991 99 95 47 39 Cycle Number Threshold Set Too Low The threshold is set below the exponential phase of the amplification curve The standard error 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 Delta Rn Sample Detector Task Liver CD30 Target 21 117 0 006 0 246 0 000 1 000 1 000 0 659 1 517 0 Kidney CD30 Target 21 5141 688 0 264 1 694 0 309 0 002 0198 0483 oO g Rees OESE EEES EEEE BO KEES En TE Easa Ee oc KA BPs Eee HEEE Kea A a Ee es HE Bladder CD3D Target 24 0432673 0 261 2679 0156 0 002 0 097 0 252 0 Cycle Number Threshold Set Too High The threshold is set above the exponential phase of the amplification curve The standard error 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 Delta Rin Aug Aug dct std P RO RO Outl Sample Detector Task ddt Ro Liver CD3D Target 33449412527 0414 0o00 1 000 1 000 0 824 1214 0 Kidney CD30 Target 34493 14 667 O449 2 340 0197 0 002 0 092 0 423 0 1 3 5 7 111315 17 19 21 23 25 2F 29 31 33 35 37 39 Bladder CD3D Target 3831416944 0 377 4 617 0 041 0 002
32. 0001 Linear Minimum ne L on e Log te Log Maximum 10 Maximum Axis is autoscaled in RealTime Axis V Auto Scale t Minimum Ke Pc Maximum Display Options Line Width 2 1 10 Defaults Cancel Apply 38 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Plate Tab Displays the results data of each well including the e Sample name and detector task and color for each well e Calculated R value 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 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 Analyzing and Viewing RQ Plate Data Plate Tab d EAN Y Spectra f Component Plot 6 42 B Liver 557 C Liver 6 51 D Liver 594 E Liver 675 2 Liver 6 44 Liver 593 Liver 6 53 Liver 6 09 Liver 6 96 3 Liver 6 43 Liver 595 Liver 6 91 Liver TEE Liver H70 Setup Instrument Results Plate Spectra Y Component Amplification Plot 9220 8000 6000 4000 Fluorescence 2000 228 Note If you are using Taq
33. 1 82 R reagent configurations 20 real time PCR assays 2 reference sample See calibrator reference passive 4 33 50 relative quantification comparative method of calculation 3 definition 2 example experiment 6 experiments See also RQ experiments 3 real time PCR 2 references 2 RQ plates 3 RQ studies 3 88 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System replicates 17 reporter dye 4 Results tab 38 56 results RQ Study analysis 72 reverse transcription guidelines for preparing RNA 24 High Capacity cDNA Archive kit 25 thermal cycling parameters 25 Rn vs Cycle view 73 Rn See normalized reporter RNA guidelines for preparing 24 isolation 24 starting concentration 24 ROX dye 33 39 50 57 RQ Detector grid 65 RQ experiments chemistries 19 components 17 probes and primers 21 reagent configuration 19 requirements 17 workflow 3 RQ Main Study view 63 RQ plates adding to RQ studies 63 Amplification Plot view 39 57 analyzing 38 56 Component view 39 57 datatypes 40 58 definition 3 detectors creating 81 exporting data 40 58 Plate view 39 57 reanalyzing data 40 58 results 38 56 RQ Plate documents 31 47 Spectra view 39 57 starting a run 37 55 RQ Results panel 72 RQ Sample grid 72 RQ studies adding RQ plates 63 Amplification Plots 72 confidence level 65 control type 65 data types 79 definition 3 exporting data 79 Gene Exp
34. Cycle Number PAS Foie N BEREC IC estos fan a Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Detector Al z Line Color well Color z 39 Chapter 4 Generating Data from RQ Plates 7300 or Standard 7500 System Exporting RQ Plate Data Reanalyzing Data Raw fluorescence data spectra R values and well information sample name detector and detector task are saved in an RQ plate document If you decide to omit wells or change well information after a run is complete you must reanalyze the data Note After the software analyzes data the Analyze button is disabled Whenever you change a setting that requires reanalysis the Analyze button is enabled Exporting RQ Plate Data You can export numeric data from RQ plates into text files which can then be imported into spreadsheet applications such as Microsoft Excel 1 Select File gt Export then select the data type to GDN View Tools Instrument Analysis Window Help export Mew Ctrl M Open Chrl o e Sample Setup txt Close Save Chrl 5 e Calibration Data csv Save As xk Import Sample Setup e Spectra csv TEER Component csv view Exported Results Calibration Data Page Setup Spectra e Rn csv Print Preview Component Print Ctrl F RD Typically you export sample setup data for Te newly created and newly run plates other
35. Man products three components ROX dye reporter dye and TAMRA quencher are displayed in the Component tab If you are using TagMan MGB products only two components ROX and reporter dyes are displayed as shown in the figure on the right Amplification Plot Tab Displays a plot of R as a function of cycle number for the selected detector and well s Notes Liver 6 66 Liver 597 Liver 6 71 Liver 6 22 Liver 731 E z se s wj Liver 5 34 Liver T 652 Liver MER Liver 7 01 Liver T 548 Liver 531 Liver T 6 28 Liver iT 4 34 Liver 6 94 Liver T 545 Liver 5 23 Liver T 6 36 Liver 7 470 Liver 6 97 Liver T540 Raw Data Liver fa 5 10 Liver T672 Liver M476 Liver 7 00 Liver T 514 Liver 7 83 Liver H 2 46 Liver T 6 38 Liver 6 85 Liver TERN Liver rs Liver M259 Liver Tsis Liver Hes Liver H i Setup Instrument Y Results Plate Spectra Y Component Amplification Plot Component Sa BEE eo ee ee 3200 00 2800 00 2400 00 2000 00 1600 00 Fluorescence 1200 00 800 00 400 00 Cycles Component s Mv Fam WY Rox Setup Instrument Results Plate Spectra Component Amplification Plot Rn vs Cycle Sasa Ua base HES Feet Hace M denM Bea A Pepe am O Ceara
36. Note The SDS software uses this value to calculate error bars for gene expression levels as explained in Error Bars for Gene Expression Plots on page 75 8 Optionally select Remove Outliers to enable the SDS software to automatically identify and filter outliers for groups containing at least four replicates Note You can also remove outliers manually as explained in Omitting Samples from a Study on page 77 9 Click OK amp Reanalyze The detector Y Amplification Plat Y Gene Expression information appears in the RQ Detector grid Detector Threshold AutoCT Baseline A me E AE Last Moaea ACYR1 0 535276 Auto Auto Kidney a 05 2003 02 After analysis the Threshold column displays eee AS Ae ae Liver Aug 05 2003 02 the automatically calculated threshold values Sooo fe spay tae ae FLT4 0 451420 Auto Auto The Auto Ct and Baseline columns are set to sappa a tka a oA t 99 GTF2B 0 251276 Auto Auto uto GTF 2l 0 280024 Auto Auto HTR4 0 082686 Auto Auto 5 1 1 KAI 0 323054 Auto Auto For more information about the settings in the a paos Aado Auto Analysis Settings dialog box refer to the Online a e fo Ato 3 Help Se aaa s s Eind a E Sample Summary Well Information Add Plate Remove Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 65 SSS Chapter 6 Analyzi
37. OX dye is selected d Repeat steps a through c until you specify sample names and passive reference dyes for all the wells on the plate Sample Hame Liver Detector Repor Reporter Quencher Task Color eae i ly RARA FAN fone Target RARG FAM Target IMPORTANT If your experiment does not Cre Ta f menes Tarom use all the wells on a plate do not omit the M TAcR2 FAM none Target i E ACR FAM none Target wells from use at this point You can omit rere a Omit well unused wells after the run For information about omitting unused wells refer to the Online Help Add Detector Remove Close Note You can change the sample setup information sample name detector task after a run is complete if necessary e Close the Well Inspector Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 33 Chapter 4 Generating Data from RQ Plates 7300 or Standard 7500 System Creating a Relative Quantification RQ Plate Document 9 Verify the information on each well in the Setup tab Example Experiment In the example RQ experiment the samples for each of the three tissues liver kidney and bladder are loaded on three separate plates Consequently three RQ Plate documents are created one for each of the sample plates Because the experiment is singleplex there
38. PORTANT The protocol is not shipped with the High Capacity cDNA Archive 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 Protocols heading Thermal Profile The High Capacity cDNA Archive Kit uses the following thermal profile parameters for Parameters for RT the RT step Step Type Time Temperature HOLD 10 min 25 C HOLD 120 min 37 C Note Thermal cycling conditions for one step RT PCR are described on page 35 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 25 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 Nae CHEMICAL HAZARD 10 x RT 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 RNA is extracted from the liver bladder and kidney tissues of an individual RNA concentration is determined spectrophotometrically using Ago and t
39. R Systems Chemistry Guide PN 4348358 ABI PrisM 7700 Sequence Detection System User Bulletin 2 Relative Quantitation of Gene Expression PN 4303859 How to Obtain Services and Support 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 Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Vill Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Send Us Your Comments Safety Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Ix Preface Send Us Your Comments X Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Introduction and Example RQ
40. SDS software will reject a plate 1f it detects any differences The first plate added to the study serves as the reference plate against which other plates are compared o a ee i Remove 6 Click Finish If desired save the RQ Study feb S6niae E Omit Det Deta PRnvs Cycle _ Calit document when prompted fc nC PlateHame Last Modified The SDS software opens a new RQ Study _ Detector Threshold Auto CT eee Rae cater f ACYRI 0200000 Manual l document and displays the RQ Study Main view ACVR2 0200000 wea 2 oey ii T i ug 05 with its three panes Sa cap Manus CDS ALLELE Manual a FisF21 0 200000 Manual a RQ Detector grid Allows you to select e F aaeaaal e detectors to associate with the loaded study SAn Se For each detector Color Detector name GTFal 0200000 Manual HTR4 0 200000 Manual Threshold value Auto Ct and Baseline are re 9200000 Manual MCAT 0 200000 Manual displayed MFA 0 200000 Manual PT 4 AEE L H o gt Note At this point all the values in the aa Threshold Auto Ct and Baseline columns Eind zj are set to the default values 0 200000 E log Manual and 6 15 respectively a 4 ii b RQ Sample grid Displays the samples Sample Summary ee Add Plate Remove m associated with the selected detector s The Sample Grid displays numerical results of RQ calculations and has two subtabs Sample Summary and Well Information
41. Template Blank Document parameters the same number of steps l cycles sample volume emulation mode poe The SDS software will reject a plate if it Operator Administrator detects any differences The first plate added to the study serves as the reference plate against which other plates are Default estas Plate Mame Paas Comments a Select File gt New os Finish __ Cance Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 9 Chapter 1 Introduction and Example RQ Experiment Example RQ Experiment b Select Relative Quantification ddCt Plate in the Assay drop down list then click Next gt IMPORTANT You cannot use AQ Plate documents for RQ assays and vice versa The information stored in AQ and RQ Plate documents is not interchangeable You cannot import an AQ plate into an RQ study You cannot analyze your relative expression data directly in an RQ plate setup You cannot run a standard curve or a dissocication curve in an RQ plate setup You can only analyze relative expression data in an RQ study setup c Add detectors to the plate document then click Next gt d Specify the detectors and tasks for each well then click Finish You cannot add RQ plates to RQ studies unless you have specified sample names as indicated in the message shown to the right Click OK in place s
42. Well Inspector Creating an RQ Plate Document New Document Wizard Select Detectors Select the detectors you will be using in the document Detector Nar 1e Quenche Detectors in Document FAM none GAPDH FLT CD3D Remove New Detector lt Back Next gt Finish Cancel Hew Document Wizard x Echup Sample Plate Setup che sampl pace mth tasts quart tec and detectire uea Detector Reparar Guencher loak coor Tara Tab tn n Tater i ACY FAN none Target 2 BrE inc n AME Tah eS i a Target Larg ENCO a 4 s r wfliw 2 aif ed al b bid ba Bark Eaa l cere SDS v1 3 Note You will need to enter sample names before sawing the RO Plate document You can enter sample names by using the Well Inspector or by using the in place sample name editing Feature select wellis and type text directly into the sample name After editing sample name s you can then save the RO Plate document Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 49 Chapter 5 Generating Data from RQ Plates 7500 Fast System Creating a Relative Quantification RQ Plate Document 8 Enter the sample names a In the Well Inspector click a well or click drag to select replicate wells b Enter the sample name c If necessary c
43. ample name editing feature select wellis and type text directly into the sample name Note You will need to enter sample names before saving the RQ Plate document You can enter sample names by using the Well Inspector or by using the After editing sample name s you can then save the RQ Plate document Notes 10 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Example RQ Experiment Example RQ Experiment Procedure The SDS software displays the Well Inspector RNase P FAM none Unknown Omit Well Passive Reference Add Detector Remove Close ROS r 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 is completed For more information about omitting wells refer to the Online Help The figure on the right shows a completed plate Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System set up Seto Fist en Frears Y F Piste OLA ae a s u w a x A uer Lee Uver Lke Uver Lee Uver Lee Uver Lee Uver Lie H E B uwer Lee Over Lee Uver Lee uer Lker er Lee Uver Lee z 5 Z Uvar Lhe Uver Uver Lhe Uvar Ler Uvar Ler Uvar Ler E E D uwer Lee
44. an use this plot to locate outliers from detector data sets see Omitting Samples from a Study on page 77 for more information Note If there is a data point at Ct0 this point is undetermined the data point is not actually Ct 0 Notes Analyzing and Viewing the Results of the RQ Study Amplification Plot fe Omit Data Rn vs Cycle Calibrator Liver Plate Amplification Plot Gene Expression 9 5 7 8 11 19 48 47 18 21 Bi 26 27 2a St 39 oe ar a Cycle Number 1 0e 001 1 0e 000 1 0e 001 Delta Rn 1 0e 002 E 1 0e 003 ae 1 0e 004 1 3 5 7 9 1113 15 17 19 21 23 25 27 29 31 33 35 37 39 Cycle Number e Omit Data Ctvs Well Position Calibrator Liver Orie Amplification Plot Gene Expression Ct vs Well Position Ct 0 1 5 9 1317 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 Well Position Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 73 Chapter 6 Analyzing Data in an RQ Study Analyzing and Viewing the Results of the RQ Study Gene Expression Plot Gene Expression plots show the expression level or fold difference of the target sample relative to the calibrator Because the calibrator is compared to itself the expression level for the calibrator is always 1 Adjusting Graph Settings
45. annot use RQ Plate documents for AQ assays and vice versa You e T cannot use the RQ Plate to perform relative quantification using the relative standard curve method The information stored in AQ and RQ Plate documents is not interchangeable 4 Enter a name in the Default Plate Name field or accept the default 5 Click Next gt Notes 48 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Creating a Relative Quantification RQ Plate Document 6 Select detectors to add to the plate document a Click to select a detector Ctrl click to select multiple detectors If no detectors are listed in the Detector Manager create detectors as explained in Appendix A Creating Detectors on page 81 Click Add gt gt The detectors are added to the plate document Note To remove a detector from the Detectors in Document panel select the detector then click Remove Click Next gt T Specify the detectors and tasks for each well Notes a Click a well or group of wells for replicates to select it Click to select the detector s for the well Click under the Task column to assign the detector task Select Use Click Finish You cannot add RQ plates to RQ studies unless you have specified sample names as indicated in the message shown to the right Click OK The SDS software creates the plate document and displays the
46. ative Quantification RQ Plate Document Overview Creating a Relative Quantification RQ Plate Document Overview Run Setup Requirements Notes An RQ Plate document stores data collected from an RQ run for a single plate There must be one RQ Plate document for every RQ plate RQ Plate documents also store other information including sample names and detectors For each RQ plate document that you create specify detectors endogenous controls and detector 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 sequence Appendix A explains how to create detectors IMPORTANT To conduct a comparative analysis of the data in a study all the plates in the study must contain a common set of detectors An endogenous control s is defined in Specifying the Components of an RQ Experiment on page 17 If your experiment consists of multiple plates each plate must have at least one endogenous control with at least three replicates If your experiment consists of a single plate with multiple samples there must be an endogenous control for each sample All plates in an RQ experiment must use the same endogenous control for example GAPDH A detector task specifies how the software uses the data collected from the well during analysis You apply one of two tasks to each detector in each well of a plate document
47. ave already completed the RT step and are ready to PCR amplify cDNA A AddCycle Add Hold Add Step Add Dissociation Stage Help The default thermal cycling conditions for the PCR ee amy E fanmode SEE A Sample Volume pL un Mode Standar step of the procedure shown in the following table O TOA should appear in the Instrument tab Times and Temperatures Two step RT PCR HOLD HOLD For reference only RT is complete at this 1 RT Step point 10 min 25 C 120 min 37 C Initial Steps PCR Each of 40 cycles AmpErase UNG AmpliTaq Gold DNA Melt Anneal Extend 2 PCR Step Activation Polymerase Activation HOLD HOLD CYCLE 2 min 50 C 10 min 95 C 15 sec 95 C 1 min 60 C Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 35 Chapter 4 Generating Data from RQ Plates 7300 or Standard 7500 System Specifying Thermal Cycling Conditions and Starting the Run Thermal Cycling Conditions for One Step RT PCR If you select the one step RT PCR method cDNA Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate generation and amplification take place a eee ees rene 1 Rees E Fens simultaneously at this point in the workflow The following table shows the thermal cycling conditions for one step RT PCR experiments Add Dissociation Stage
48. ck Start After the run a message indicates if the run is successful or if errors were encountered Create an RQ Study document as described in Creating an RQ Study Document on page 62 Hew Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Relative Quantification ddCt Study Y 96 ell Clear r paben o a Select File gt New b Select Relative Quantification ddCt Study in the Assay drop down list then click Next gt Container Template Operator Administrator ne Default Plate Name Plate1 Finish Cancel IMPORTANT RQ Study is an optional add on for the 7300 instrument it 1s built in for the 7500 instrument and the 7500 Fast instrument Comments c Click Add Plates to add plates to the study then click Open Note You can add up to 10 RQ plates to an RQ study d Click Finish Notes 12 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Example RQ Experiment Conclusion 10 Analyze the RQ data as explained in Chapter 6 Analysis Settings Relative Quantification on page 61 Ct Analysis a Configure analysis settings m using the ree E pi f Auto Ct Auto Ct option and analyze the data C Manual Ct Note See Configuring Analysis Settings Theshol 0 200000 on page 64
49. commended you have already completed the RT step and are ready to PCR amplify m Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 cDNA Reps Reps The default thermal cycling conditions for the PCR step of the procedure shown in the following table should appear in the Instrument tab Fast Default Times and Temperatures Two step RT PCR HOLD HOLD For reference only RT is complete at this 1 RT Step l 10 min 25 C 120 min 37 C paint Fast Thermal Cycling Conditions Fast 7500 users only Enzyme Activation Melt Anneal Extend 2 PCR Step i 95 C 3 sec 95 C 30 sec 60 C Fast Conditions 0 20 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 53 Chapter 5 Generating Data from RQ Plates 7500 Fast System Specifying Thermal Cycling Conditions and Starting the Run To specify thermal cycling conditions and start the run 1 Select the Instrument tab Ka 7500 Fast System SDS Software Plate1 Relative Quantification Plate By default the standard PCR conditions for the ee PCR step of the two step RT PCR method are Instrument Control Temperature f start Estimated Time Remaining hh mm Sample Heat Sink displayed Cover Block Status Stage Rep Time mm ss Step 2 Verify that Thermal Cycler Protocol
50. curve or gel analysis fluorescence detected by the instrument Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 19 Chapter 2 Designing an RQ 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 ina single reaction one step or in separate reactions two step The reagent configuration you use depends on whether you are performing one step or two 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 uracil N glycosylase enzyme can be used to prevent carryover contamination IMPORTANT This guide assumes that RQ experiments are designed using two step RT PCR For additional options refer to the SDS Chemistry Guide Two Step RT PCR IMPORTANT RQ plates run standard thermal cycling conditions Fast thermal cycling conditions cannot be combined into a single RQ study e 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 cannot use the carryover p
51. e Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 79 Chapter 6 Analyzing Data in an RQ Study Exporting RQ Study Data Notes 80 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Creating Detectors 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 Select File gt New In the 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 Optionally click the Description field then enter a brief description of the detector Notes Detector Manager 20050915 05 20050815 05 2005097350 13 20050930 13 200509350 13 20030850 13 20030915 05 ANN oR 1A be none RARA FAM none RNase F 3 FAM none RNase F 2 FAM none RNase F 1 FAM none RNase F FAM none KAM FAM none IP Meterctor Internal Positive tli nanet
52. e Expression Assays e Applied Biosystems Web site predesigned primers and probes e TaqMan Custom Gene Expression Assays service predesigned primers and e Contact your Applied Biosystems Sales probes Representative e Primer Express Software custom designed primers and probes Reagent tubes with caps 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 Safety goggles MLS Vortexer MLS Notes 5 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Chapter 1 Introduction and Example RQ Experiment Example RQ Experiment Example RQ Experiment Overview To better illustrate how to design perform and analyze RQ experiments this section guides you through an example experiment The example experiment represents a typical RQ experiment setup that you can use as a quick start procedure to familiarize yourself with the RQ workflow Detailed steps in the RQ workflow are described in the subsequent chapters of this guide Also in the subsequent chapters are Example Experiment boxes that provide details for some of the related steps in the example experiment Description The objective of the example RQ
53. e Fold expression changes are calculated using the equation 2 407 J ea amp 6 E E Omit Data Delta Rnvs Cycle v Calibrator Liver Orientation Detector Find al 7 Plate Amplification Plot Gene Expression 0 20 I Log 10 Relative Quantification 1 20 Z 1 40 Bladder CD3D Target 31 909 N N a bl I z e e 5 a a a gi gt Q Q oO O ca a Sample Summary Well Information Detectors Notes 72 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Amplification Plot The three Amplification Plots allow you to view post run amplification of specific samples The Amplification Plots display all samples for selected detectors You can adjust graph settings by double clicking the y or x axes of a plot to display the Graph Settings dialog as shown on page 38 Rn vs Cycle Linear View Displays normalized reporter dye fluorescence R as a function of cycle You can use this plot to identify and examine irregular amplification For more information about R refer to the SDS Chemistry Guide 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 parameters for the run Ct vs Well Position View Displays threshold cycle Cy as a function of well position You c
54. e Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Primer Extended on mRNA 5 Performing Reverse Transcription Oligo Synthesis of 1st cDNA strand ai Workflow Performing Reverse Transcription Isolate total RNA See page 24 Adjust RNA 24 concentration zas PELE Convert 7 total RNA to cDNA ee page 25 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 23 Chapter 3 Performing Reverse Transcription Guidelines for Preparing RNA Guidelines for Preparing RNA Isolating RNA Applied Biosystems supplies several instrument systems and chemistries for RNA isolation from a variety of starting materials such as blood tissue cell cultures and plant material Products Part Number ABI PrisM 6100 Nucleic Acid PrepStation 6100 01 Total RNA Chemistry Reagents Nucleic Acid Purification Elution Solution 4305893 Nucleic Acid Purification Lysis Solution 4305895 Nucleic Acid Purification Wash Solution 4305891 Nucleic Acid Purification Wash Solution II 4305890 AbsoluteRNA Wash Solution DNase treatment 4305545 Tempus Blood RNA Tubes 4342972 For collection stabilization and isolation of total RNA in whole blood for gene expression analysis using the 6100 PrepStation Isolation of Total RNA from Whole Blood and from Cells Isolated from 4332809 Who
55. e00060060060080 80 eee0000600080 80 SISISI SISOS G18IST Qc SISIGIG CISION GIS pies 2 ee eee0006006000 80 eee000000680 80 2 eeee00000 0 0 Endogenous 600000000009 OF controls GAPDH Notes 18 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Selecting the Chemistry About Chemistries 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 SDS Chemistry Guide Chemistry Process TaqMan reagents or kits Polymerization Strand Displacement Description Qmo Q a ongen a TaqMan reagent based chemistry uses a re ee H y fluorogenic probe to enable detection of a specific ae 5 3 PCR product as it accumulates during PCR cycles Step 1 A reporter R and a Step 1 continued When both dyes Advantages quencher Q are attached to the are attached to the probe reporter e Increases specificity with a probe Specific 5 and 3 ends of a TaqMan dye emission is quenched hybridization between probe and target probe generates fluorescence signal e Provides multiplex capability
56. ected well over the duration of the PCR run Only the first selected well is shown at one time Analyzing and Viewing RQ Plate Data Plate Tab d EAN Y Spectra f Component Plot 6 42 B Liver 557 C Liver 6 51 D Liver 594 E Liver 675 2 Liver 6 44 Liver 593 Liver 6 53 Liver 6 09 Liver 6 96 3 Liver 6 43 Liver 595 Liver 6 91 Liver TEE Liver H70 Setup Instrument Results Plate Spectra Y Component Amplification Plot 9220 8000 6000 4000 Fluorescence 2000 228 Note If you are using TaqMan products three components ROX dye reporter dye and TAMRA quencher are displayed in the Component tab If you are using TagMan MGB products only two components ROX and reporter dyes are displayed as shown in the figure on the right Amplification Plot Tab Displays a plot of R as a function of cycle number for the selected detector and well s Notes Liver 6 66 Liver 597 Liver 6 71 Liver 6 22 Liver 731 E z se s wj Liver 5 34 Liver T 652 Liver MER Liver 7 01 Liver T 548 Liver 531 Liver T 6 28 Liver iT 4 34 Liver 6 94 Liver T 545 Liver 5 23 Liver T 6 36 Liver 7 470 Liver 6 97 Liver T540 Raw Data Liver fa 5 10 Liver T672 Liver M476 Liver
57. ed Biosystems 7300 7500 7500 Fast Real Time PCR System Chapter 1 Introduction and Example RQ Experiment About RQ Experiments About RQ Experiments RQ Experiment Workflow RQ Studies with the 7300 7500 7500 Fast System Terms Used in Quantification Analysis Notes In this document the term RQ experiment refers to the entire process of relative quantification beginning with generating cDNA from RNA reverse transcription and ending with analyzing an RQ study The RQ experiment workflow is shown on page iil The data collection part of an RQ assay is a single plate document called the RQ Plate Amplification data from PCR runs is stored with sample setup information on the plate The data analysis part of an RQ assay is a multi plate document called the RQ Study You can analyze up to ten RQ plates in a study RQ Study documents neither control the instrument nor do they provide tools for setting up or modifying plates IMPORTANT RQ Study software is an optional package for the 7300 instrument but is standard for the 7500 instrument and the 7500 Fast instrument The following figure illustrates the RQ Study process il i ay i kidneyplate sds bladderplate sd Plated Reactions 7300 7500 7500 Fast System SDS v1 3 Software RQ Plate Documents SDS v1 3 Software RQ Study Document Note The 7300 7500 7500 Fast system software uses the comparative method AAC to calculate relative
58. eee eee nM aa ahaate amp 15 selecting the PCR MEtNOd resar keel bide ee en cet eee bated bea 16 Specifying the Components of an RQ Experiment 2 0 00 cee eee ees 17 Selecting the Chemistry cesser rinner e E eee eee teens 19 Selecting One or Two Step RT PCR 2 0 tees 20 Choosing the Probes and Primers 0 0 00 ccc eee eee ete eens 21 Chapter 3 Performing Reverse Transcription 23 NVVOKKTIOW eine aa we a Al a ae lee ea ee ee ee ee eA hae Eee ks Mae Rens 23 Guidelines for Preparing RNA 000 cc eee eee ees 24 Converting Total RNA to CDNA 0 00 cc tees 25 Chapter 4 Generating Data from RQ Plates 7300 or Standard 7500 System 27 WORK TOW see Mesh S das aerial Roe teeth nea Wed ar ie eared tale dt Se ead tak etee O A 27 Before YOU BEIN erus harie how as ae ie eh a boc N a E 28 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System V Preparing the PCR Master Mix nananana 600d geawd stg eukeod owe twee ae ds 28 Preparing the Reaction Plate 0 cc eee 29 Creating a Relative Quantification RQ Plate Document 0000 0 ee 31 Specifying Thermal Cycling Conditions and Starting the Run 35 Analyzing and Viewing RQ Plate Data 0 0 ccc eee 38 Exporing R Plate Dalas eier iA dash e O48 ew de A he eee oe ae ewe ok 40 Chapter5 Generating Data from RQ Plates 7500 Fast System 41 WOKO sata Gig
59. en views click a tab e To select all 96 wells on a plate click the upper left corner of the plate e To adjust graph settings double click the y or x axes of a plot to display the Graph Settings dialog The adjustable settings depend on which plot you are viewing Notes F Fie view Tools Instrument Analysis Window Help Bp lA Blo E setup Y instrument Yresuts Y 7 Plate Spectra Y Amplification Plot B Liver Liver Liver Liver ia Liver 5 87 5 95 5 95 5 97 6 52 6 265 Graph Settings Real Time Settings Post Run Settings Y Axis Auto Scale Minimums Y Axis V Auto Scale C Linear Minimum 0 0001 Linear Minimum ne L on e Log te Log Maximum 10 Maximum Axis is autoscaled in RealTime Axis V Auto Scale t Minimum Ke Pc Maximum Display Options Line Width 2 1 10 Defaults Cancel Apply 56 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Plate Tab Displays the results data of each well including the e Sample name and detector task and color for each well e Calculated R value 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 Component Tab Displays the complete spectral contribution of each dye in a Sel
60. ent a detector is created for each target gene and the endogenous control 24 detectors are created 23 for the target genes and 1 for the endogenous control GAPDH For example the detector for the ACVR1 gene is named ACVR1 and assigned a yellow color Because all TaqMan Gene Expression Assays have probes that are labeled with FAM dye FAM was selected for the reporter dye Additionally TaqMan Custom Gene Expression Assays use TaqMan MGB probes which use nonfluorescent quenchers None is selected for the quencher dye Notes 82 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Note TaqMan Genotyping 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 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 83 Appendix A Notes 84 Relative Quantification Getti
61. ents you can use the same dye for targets and endogenous control s For multiplex experiments the probe for the target is typically labeled with FAM dye and that for the endogenous control with VIC dye Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 21 Chapter 2 Designing an RQ Experiment Choosing the Probes and Primers Example Experiment For the example experiment primers and probes for all the genes being studied are obtained from Applied Biosystems TaqMan Gene Expression Assays Each assay consists of two unlabeled PCR primers and a FAM dye labeled TaqMan MGB probe provided as a 20X assay mix In the example experiment all target probes are labeled with FAM dye the endogenous control is also labeled with FAM dye The following table provides the gene symbol gene name and Applied Biosystems Assay ID number provided on the Web site for five of the genes studied in the example experiment plus the endogenous control Gene Symbol Gene Name Assay ID ACVR1 acrosomal vesicle protein Hs00153836 m1 ACVR2 activin A receptor type Il Hs00155658_m1 CCR2 chemokine C C motif receptor 2 Hs00174150_m1 CD3D CD3D antigen delta polypeptide TiT3 complex Hs00174158_m1 FLT4 fms related tyrosine kinase 4 Hs00176607_m1 GAPDH glyceraldehyde 3 phosphate dehydrogenase Hs99999905_m1 Notes 22 Relativ
62. ermal Cycling Conditions for One Step RT PCR e 9600 Emulation is selected Note If you are using SYBR Green I reagent chemistry and you want to determine if there is contamination or if you want to determine the dissociation temperature create a separate Dissociation assay or template Refer to the Online Help for more information Note In the 7300 instrument the 9600 Emulation feature is not available 3 Select File gt Save As enter a name for the RQ Plate document then click Save 4 Load the plate into the instrument Note 7300 and 7500 standard plates are notched Well A1 in the A12 position at the top right Fast plates are notched in the A1 position at the top left 5 Click Start Keyed As the instrument performs the PCR run it corner for displays real time status information in the gale Instrument tab and records the fluorescence emissions After the run a message indicates whether or not the run is successful All data generated during the run are saved to the RQ Plate document that you specified in step 3 Keyed corner for Fast plates Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 37 DA RD Analyzing and Viewing RQ Plate Data Chapter 4 Generating Data from RQ Plates 7300 or Standard 7500 System Analyzing and
63. everse primers and labeled probe b 24 master mixes are prepared one for each of 23 genes plus the endogenous control Volume for five reactions 4 replicates plus extra i uL a Reaction Component Sample uL 5 Reactions Final Concentration TaqMan Fast Universal PCR Master Mix 10 50 1X 2X 20X TaqMan Gene Expression Assay 1 5 1X Mix cDNA sample 50 ng for the 50 uL reaction 9 45 Nuclease free water Total 20 100 Samples and endogenous controls are arranged on three plates as shown below 20 uL of Fast PCR master mix containing Bladder samples 000000 eeeeee ee eeeee FILII ee eeeee eee0e0e0 ee eeeee FIRII O O QOeeeoeee The reactions are kept on ice until the plates are loaded on the 7500 Fast system Endogenous controls GAPDH Note 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 Notes 46 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Creating a Relative Quantification RQ Plate Document Overview Creating a Relative Quantification RQ Plate Document Overview An RQ Plate document stores data collected fro
64. ferent from the target wells Four replicates of each sample and endogenous control are performed to ensure statistical significance See below Note The example RQ experiment requires a separate plate for each of the three tissues because of the large number of genes being studied You can also design experiments so that several samples are amplified on the same plate as shown in the following table In the example RQ experiment each plate contains a If the example experiment were run with multiple sample single sample type tissue The endogenous control for types on the same plate an endogenous control for each each tissue is on the same plate as the targets for that sample type must also be included on the same plate as tissue shown here Liver Kidney Bladder e0060e08080600008 ooo ooo o o o o 2e e0e000000600680 2 e eee000000090 eeeeeeeooeeee 0080 08008000 00000000P samples GIGIO CIOTOTO SOOO Samples 2 eee0000000 80 2 ee0020000090 2 eee0000000 0 e ee00000090 SISIGIS GISISIS SISISIO dogenocus eoeeee0e0e000e SIGIOIO GIOIOIO O O O controls GAPDH O00 0G CO 0CG OO GD Endogenous controls GAPDH 2 ee eee000080 80 0 EEE EEEEEEEE EEKEREN EEJ EEE Kidne eeeeeeeeecee 7 0000808000000 P 2 e ee0000080 GISISTO GISIGIS SISIGIO Er dogenous 10 O O OOOO OO OF controls GAPDH ee
65. fication Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Chapter 2 Designing an RQ Experiment Workflow Notes Designing an RQ Experiment Select the PCR method See page 16 me 1 Specify the components of an EIERN RQ experiment Select the chemistry See page 19 Select one step or See page 20 two step RT PCR Choose the probes See page 21 and primers Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 15 Chapter 2 Designing an RQ Experiment Selecting the PCR Method Selecting the PCR Method Types of PCR PCR is performed as either of the following Methods e A singleplex reaction where a single primer pair is present in the reaction tube or well Only one target sequence or endogenous control can be amplified per reaction e A multiplex reaction where two or more primer pairs are present in the reaction Each primer pair amplifies either a target sequence or an endogenous control LI m M Target Primer Set L OO O Endogenous Control VW VW Dg Primer Set l CDNA Singleplex PCR Multiplex PCR Selection Criteria Both methods give equivalent results for relative quantification experiments To select a method consider the e Type of chemistry you use to detect PCR products Singleplex PCR can use either SYBR Green o
66. hange the setting for the Passive Reference dye By default the ROX dye is selected ell Inspector Wells 41 44 d Repeat steps a through c until you specify sample names and passive reference dyes for all the wells on the plate Sample Name Liver er Quencher Task Color a Use Detector Repor RARA FAN fone Target RARG FAM Target IMPORTANT If your experiment does not RRS Tam j menes Tarom use all the wells on a plate do not omit the g TACR2 FAM none Target SERRI esd P kaane Target 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 Orit well Add Detector Remove Note You can change the sample setup information sample name detector task after a run is complete if necessary e Close the Well Inspector 9 Verify the information on each well in the Setup tab Notes 50 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Creating a Relative Quantification RQ Plate Document Creating an RQ Plate Document Example Experiment In the example RQ experiment the samples for each of the three tissues liver kidney and bladder are loaded on three separate plates Consequently three RQ Plate documents are created one for each of the sample plates Because the e
67. he RNA is diluted to a final concentration of 50 ng uL The RT master mix is prepared as follows using guidelines from the High Capacity cDNA Archive Kit Protocol Component uL Reaction uL 21 Reactions 10X Reverse Transcription Buffer 10 210 25X dNTPs 4 84 10X random primers 10 210 MultiScribe Reverse Transcriptase 50 5 105 U uL Nuclease free water 21 441 Total per reaction 50 1050 a Each RT reaction is 100 uL see below If you need 5 uL cDNA for each of 104 PCR reactions per tissue see Creating a Relative Quantification RQ Plate Document on page 31 you need 6 RT reactions per tissue Extra volume enough for one additional RT reaction per tissue is included to account for pipetting losses as well as extra cDNA for archiving The cDNA archive plate is then prepared by pipetting into each well e 50 uL ofthe RT master mix e 30 uL of nuclease free water e 20 uL of RNA sample bringing the total starting amount of RNA to 1 ug per 100 uL reaction Liver Kidney Bladder a woes beeueu ce E E OE EE EREE ES e e2ee 6 000080 86 eee2eaee8 e000 86 e2e2eee eee e 6 ee2eeoeo eee ed 6 EEKEREN e2e2eea ee ee 8 E The RNA is then converted to cDNA using the thermal cycling parameters for two step RI PCR as described in Thermal Profile Parameters for RT on page 25 The cDNA is stored at 20 C until use Notes 26
68. hemical N or Vente 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 Neon Indicates a potentially hazardous situation that if not avoided could result in death or serious injury Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System vil Preface How to Obtain More Information Safety Refer to the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Getting Started Guide and the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Site Preparation Guide for important safety information How to Obtain More Information For more information about using the 7300 7500 7500 Fast system refer to Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Online Help Applied Biosystems 7300 7500 Real Time PCR System Allelic Discrimination Getting Started Guide PN 4347822 Applied Biosystems 7300 7500 Real Time PCR System Plus Minus Getting Started Guide PN 4347821 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Absolute Quantification Getting Started Guide PN 4347825 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Getting Started Guide PN 4347828 Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Site Preparation Guide PN 4347823 Real Time PC
69. ic Avenue Alameda California 94501 USA Trademarks Applied Biosystems MicroAmp Primer Express and VIC are registered trademarks and AB Design ABI PRISM Applera Celera Genomics FAM ROX iScience iScience Design and MultiScribe are trademarks of Applera Corporation or its subsidiaries in the US and or certain 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 Microsoft and Windows are registered trademarks of Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4347824 Rev C 12 2004 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System RQ Experiment Workflow E OUOCUCNON AGOUTA About relative About Example and Example Chapter 1 HA RO E eani e ras quantification RQ experiments RQ Experiment I igni Specify the components colecti the chemisir Select one step or Choose the probes Chapter 2 eE eaerimeni Select inie FOR Metmod of an RQ experiment y two step RT PCR and primers T Performing ee Adjust RNA Convert Chapter 3 on ieee anes concentration total RNA to cDNA Generating Rad Data from Chapter 4 ANDARD RQ plates Standard Program the thermal cycling conditions Save the RQ Plate document Prepare the Prepare the Create a new i tar
70. ific amplification Normalized reporter The ratio of the fluorescence emission intensity of the reporter dye Rp 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 The figure below shows a representative amplification plot and includes some of the terms defined in the previous table Sample Threshold No Template Control Baseline 0 5 10 15 20 25 30 35 40 Cycle Number Required User Supplied Item Source Materials ABI PRismM 6100 Nucleic Acid PrepStation Applied Biosystems PN 6100 01 High Capacity cDNA Archive Kit Applied Biosystems PN 4322171 TaqMan Universal PCR Master Mix Applied Biosystems PN 4304437 TaqMan Fast Universal PCR Master Mix 2X Applied Biosystems PN 4352042 No AmpErase UNG MicroAmp Optical 96 Well Reaction Plate Applied Biosystems PN 4306757 Optical 96 Well Fast Thermal Cycling Plate Applied Biosystems PN 4346906 with Barcode code 128 Optical Adhesive Cover Applied Biosystems PN 4311971 Notes 4 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Chapter 1 Introduction and Example RQ Experiment About RQ Experiments Item Source Labeled primers and probes from one of the following sources e TaqMan Gen
71. information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 DISCLAIMER OF LICENSE No rights for any application including any in vitro diagnostic application are conveyed expressly by implication or by estoppel under any patent or patent applications claiming homogeneous or real time detection methods including patents covering such methods used in conjunction with the PCR process or other amplification processes The 5 nuclease detection assay and certain other homogeneous or real time amplification and detection methods are covered by United States Patent Nos 5 210 015 5 487 972 5 804 375 and 5 994 056 owned by Roche Molecular Systems Inc by corresponding patents and patent applications outside the United States owned by F Hoffmann La Roche Ltd and by United States Patent Nos 5 538 848 and 6 030 787 and corresponding patents and patent applications outside the United States owned by Applera Corporation Purchase of this instrument conveys no license or right under the foregoing patents Use of these and other patented processes in conjunction with the PCR process requires a license For information on obtaining licenses contact the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or The Licensing Department Roche Molecular Systems Inc 1145 Atlant
72. into the standard block correctly and will result in loss of data Fast Plates Standard Plates Standard plate has notch at top right corner by A12 A c Dod J C GD e jog D o d gt o gt a s gt yi S Fast plate has notch at top left corner by A1 FTT ust TUT UY anata reaction reaction volume volume 1 Label the reaction plates ensuring that you include an endogenous control for each sample type for example each tissue in a study comparing multiple tissues If samples are spread across multiple plates each plate must have an endogenous control Additionally every plate must include an endogenous control for every sample type on the plate 2 Into each well of the reaction plate add 50 uL of the appropriate PCR master mix 3 Keep the reaction plates on ice until you are ready to load them into the 7300 7500 system Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 29 Example Experiment Primers and probes for the example RQ experiment are obtained from the TaqMan Gene Expression Assays product line and are provided as a 20X Gene Expression Assay Mix The PCR master mix is prepared as follows Chapter 4 Generating Data from RQ Plates 7300 or Standard 7500 System Preparing the Reaction Plate Reaction Component aie a uL 5 Reactions Final Concentration TaqMan
73. ion 17 for RQ plates 31 47 selecting for RQ studies 65 endpoint PCR 2 equipment 4 example RQ experiment components 18 creating detectors 82 description 6 PCR master mix 30 46 PCR method 16 reverse transcription 26 RQ Plate document example 34 51 RQ Study document example 72 exponential phase of amplification curve 66 exporting data RQ plates 40 58 RQ studies 79 F FAM dye 21 82 Fast system creating an RQ plate document 48 example experiment 46 generating data from RQ plates 41 requirements 42 thermal cycling conditions 53 Fast vs standard plates 44 G Gene Expression plots 74 graph settings 38 56 73 74 guidelines preparing RNA 24 H High Capacity cDNA Archive kit 25 importing plate setup information 32 48 Instrument tab 36 54 L line width 38 56 73 74 linear phase of amplification curve 66 M master mix PCR 28 42 materials 4 mode emulation 37 MSDSs obtaining viii multiplex PCR 16 N New Detector dialog box 81 normalized reporter 4 O options display 38 56 73 74 options graph 38 56 73 74 outliers 77 P passive reference 4 33 50 PCR end point 2 master mix preparing 28 42 multiplex 16 real time 2 selecting a method 16 singleplex 16 starting an RQ platerun 37 55 phases of amplification curve 66 Plate view 39 57 plate RQ See RQ plates plateau phase of amplification curve 66 plot appearance 38 56 73 74 Primer Express Software 21 primers 21 probes 2
74. is included to account for pipetting losses as well as extra cDNA for archiving Liver Kidney Bladder Z 2eeee8ed 0 0 E E EOE E E E EE ES 0000o 00000 0000o 0000o 0000o 0000o 0000o EEEE E EEEE E EEEE EEEE E 0000o Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Example RQ Experiment c Program the thermal cycler using the indicated parameter values for the RT step of the two step RT PCR method Chapter 1 Introduction and Example RQ Experiment Note You have the option to use one step RT PCR as explained in Selecting One or Two Step RT PCR on page 20 Step Type Time Temperature HOLD 10 min 25 C HOLD 120 min 37 C 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 28 for more information Note The reaction volumes for TagMan Gene Expression Assays and TaqMan Custom Gene Expression Assays are specified in the product insert Those for primers and probes designed with Primer Express software follow the universal assay conditions described in Chapter 4 PCR Master Mix Standard Near CHEMICAL HAZARD TaqMan Universal PCR Master Mix may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and foll
75. is only one sample either a target or endogenous control in each well Each well is associated with a detector indicated by the colored squares Additionally each well is assigned a detector task T target or E endogenous control The figure below shows the example RQ Plate document after sample names detectors and detector tasks are assigned for each well in the liver plate 7 ard Y lamien Y sira Y Plate 1 z d 4 4 i i Hl 1U 11 12 U ar Ler U ar Ler U ar Lig Uver Ler rer L U ar Lig a Z a B Uvar Lig U ar Lig U ar Ler U ar Ler U ar Lig Uver Ler i T E m Sample name Fi i ie D uve Ler Liver Hi Lie H E a a z E Uver Lhe Uver ie lier iwe liwar iwe ar we Detector task and E E T T E a color F Uvar Leer Uver Leer Uver Leer Uvar Leer Uver Leer Uver Lhe E Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre i Uvar Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre E H El T E fl LE El EJ Notes 34 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Specifying Thermal Cycling Conditions and Starting the Run Default Thermal Cycling Conditions for PCR Specifying Thermal Cycling Conditions and Starting the Run Default Thermal Cycling Conditions for PEL PCR a Reps 7 Reps Reps If you selected the two step RT PCR method for your RQ experiment recommended you h
76. ists the universal assay conditions volume and final concentration for using the master mix Nem CHEMICAL HAZARD TaqMan Universal PCR Master Mix 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 Universal Conditions TaqMan Universal PCR 25 0 1X 1X Master Mix 2X Forward primer 5 0 50 to 900 nM 900 nM Reverse primer 5 0 50 to 900 nM 900 nM TaqMan probe 5 0 50 to 250 nM 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 All TaqMan Custom Gene Expression Assays and TaqMan Gene Expression Assays are formulated so that the final concentration of the primers and probes are within the recommended values Notes 28 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Preparing the Reaction Plate Standard vs Fast Plates Preparing the Reaction Plate Standard vs Fast Plates IMPORTANT Make sure that you use the standard Optical 96 Well Plate on the 7500 Real Time PCR system Optical 96 Well Fast Plates will not fit
77. ld settings Notes 66 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Adjusting the Baseline and Threshold Manual Baseline and Threshold Determination Baseline Set Correctly The amplification curve begins after the maximum baseline No adjustment necessary gi a wi a a GR DO A a PE By BE OO a G os DP OR a R Fe E L AE TE Cycle Number Baseline Set Too Low The amplification curve begins too far to the right of the maximum baseline Increase the End Cycle value gi Z di Baseline 357 911 1315 17 19 21 23 25 27 29 31 33 35 37 38 i Cycle Number Baseline Set Too High The amplification curve begins before the maximum baseline Decrease the End Cycle value Delta Rn Baseline Baw bear eee ee te Fake Ee be co peo fot rs a a ad ate Be in es He E Cycle Number Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 67 SSS Chapter 6 Analyzing Data in an RQ Study Adjusting the Baseline and Threshold Threshold Set Correctly The threshold is set in the exponential phase of the amplification curve Threshold settings above or below the optimum increase the standard error of the replicate groups Dalta Rn Liver Cos Target 27 492 6 369 0 111 0 000 1 0
78. le Blood Protocol Tempus Blood RNA Tube and Large Volume Consumables Protocol 4345218 Tissue RNA Isolation Isolation of Total RNA from Plant and Animal 4330252 Tissue Protocol Isolation of Total RNA from Cultural Cells ABI PRism 6700 Automated 4330254 Nucleic Acid Workstation or 6100 Nucleic Acid PrepStation Protocol Quality of RNA The total RNA you use for RQ experiments should e Have an A songo greater than 1 9 e Be intact when visualized by gel electrophoresis e Not contain RT or PCR inhibitors The High Capacity cDNA Archive Kit Protocol 4312169 contains additional guidelines for preparing the RNA template Adjusting the The High Capacity cDNA Archive Kit is optimized to convert 0 1 to 10 ug of total RNA Starting to cDNA Convert enough total RNA so that the final concentration of total RNA Concentration of converted to cDNA is 10 to 100 ng in 5 uL for each 50 uL PCR reaction Total RNA Notes OO 24 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Converting Total RNA to cDNA Using the High Capacity cDNA Archive Kit Converting Total RNA to cDNA Using the High Use the High Capacity cDNA Archive Kit PN 4322171 to perform the first step RT Capacity CDNA _ in the two step RT PCR method Follow the manual method for converting total RNA Archive Kit into cDNA as specified in the High Capacity cDNA Archive Kit Protocol PN 4322169 IM
79. les grid click to select the samples to display in the Amplification Plot Target 26 663 5 365 Target 28 406 Target 26 902 5 35 5 Verify the uniformity of each replicate population by comparing the groupings of Cy om values for the wells that make up the set TE 0 055709 0 307250 0 200000 0 352249 i G ood 0 415943 0 284137 o H 0 30520 ae clustering 0 200000 naa Ao of replicate 0 209008 ci aue data No outliers 0 421692 0 401890 0 055709 055709 0 307250 0 200000 0 352249 o 0 415943 o Potential o 0 284137 oss ad d outlier 0 200000 0 297613 0 209008 0 174955 0 350872 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 77 Chapter 6 Analyzing Data in an RQ Study Omitting Samples from a Study 6 Do one of the following e If outliers are present select the Well Information tab find the outlying sample and select the Omit check box for the sample e If outliers are not present go to step 7 T Repeat steps 5 and 6 to screen the remaining replicate groups Bladder B1 0 Bladder jec R2 37 692 0305 T 8 Select Analysis gt Analyze to reanalyze the run without the outlying data 9 Repeat steps 3 to 8 for other detectors you want to scree
80. m 17 Chapter 2 Designing an RQ Experiment Specifying the Components of an RQ Experiment Example Experiment In the example experiment the objective is to compare the expression levels of several genes in the liver kidney and bladder tissue of an individual The 23 genes of interest including ACVR1 ACVR2 CCR2 CD3D and FLT4 are the targets and the liver samples serve as the calibrator The SDS software sets gene expression levels for the calibrator samples to 1 Consequently if more ACRV1 is in the kidney than in the liver the gene expression level of ACRV1 in the kidney is greater than 1 Similarly if less CD3D is in the bladder than in the liver the gene expression level of CD3D in the bladder is less than 1 Because RQ is based on PCR the more template in a reaction the more the PCR product and the greater the fluorescence To adjust for possible differences in the amount of template added to the reaction GAPDH serves as an endogenous control Expression levels of the endogenous control are subtracted from expression levels of target genes An endogenous control is prepared for each tissue The experiment includes three sets of endogenous controls one for each tissue Also the endogenous control for each tissue must be amplified on the same plate as the target sequences for that tissue Finally note that the experiment uses the singleplex PCR format and therefore the endogenous controls are amplified in wells dif
81. m an RQ run for a single plate There must be one RQ Plate document for every RQ plate RQ Plate documents also store other information including sample names and detectors IMPORTANT RQ plates run with standard thermal cycling conditions Fast thermal cycling conditions cannot be combined into a single RQ study Run Setup For each RQ plate document that you create specify detectors endogenous controls and Requirements detector 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 sequence Appendix A explains how to create detectors IMPORTANT To conduct a comparative analysis of the data in a study all the plates in the study must contain a common set of detectors e An endogenous control s is defined in Specifying the Components of an RQ Experiment on page 17 If your experiment consists of multiple plates each plate must have at least one endogenous control with at least three replicates If your experiment consists of a single plate with multiple samples there must be an endogenous control for each sample All plates in an RQ experiment must use the same endogenous control for example GAPDH e A detector task specifies how the software uses the data collected from the well during analysis You apply one of two tasks to each detector in each well of a plate document Task Symbol Apply to detectors of
82. n Select Bladder B10 Bladder CCR2 37692 0305 fv Omit This is an enlarged version showing that the outlier is removed during analysis Notes 78 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Exporting RQ Study Data Exporting RQ Study Data You can export numeric data from RQ studies into text files which can then be imported into spreadsheet applications such as Excel 1 Select File gt Export gt Results then select the A View Tools Instrument Analysis window Help e New Ctrl M data type to export mn ne e Sample Summary csv a S p S y Save Chrl 5 e Well Information csv SANE Ai Import Sample Setup Both csv Sample Setup i view Exported Results Calibration Data Refer to the Online Help for information about Page Setup Spectra the export file types Print Preview Component Print Ctrl F Delta An Ch 1 RNAse P 2 14 05 ROStudy1 Results Sample Summary 3 Liver Well Information 4 Liver_InProgress Both 5 RgPlateTest1 6 E coli_reading 7 E coli_Plus Minus Pre Read 8 E coli_amplification QE coli_readingzinstruTab Exit 2 Enter a file name for the export file Note The name of the dialog box depends on the type of data you want to export 3 Click Save Notes Relative Quantification Getting Started Guide for th
83. nd Threshold on page 66 Alternatively you can select Manual Ct and specify the threshold and baseline manually Notes Analysis Settings Relative Quantification Ct Analysis Detector All a f Auto Ct arial Lt 3 Threshold 0 200000 a Start cycle Auto End cycle Suto Gene Expression Calibrator Sample a Endogenous Control Detector GAPDH l Control Type i Non roultiplexed ey RO Min Max Confidence 35 00 Replicate and Outlier Removal W Remove Outlier OK amp Reanalyze Cancel Apply 64 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Configuring Analysis Settings 4 Select the Calibrator Sample Note If your experiment uses only a single plate there must be at least two different samples that have different names and have their own endogenous controls You can go back to a saved RQ Plate document and change the sample names if necessary 5 Select the Endogenous Control Detector 6 Select the Control Type if the study contains both multiplex and nonmultiplex reactions Note The Multiplexed or Non Multiplexed options are active only if the plates loaded for analysis contain both multiplexed and nonmultiplexed reactions that share the same endogenous control T Select the RQ Min Max Confidence level
84. ng Data in an RQ Study Adjusting the Baseline and Threshold After the analysis you must verify that the baseline and threshold were called correctly for each detector as explained in the following section Adj usting the Baseline and Threshold Automatic Baseline and Threshold Determination The SDS software calculates baseline and threshold values for a detector based on the assumption that the data exhibit the typical amplification curve A typical amplification curve has a e Plateau phase a e Li e ee i E di eK ie SA paas b vi a a nd ave SU e Exponential geometric phase c a eae vas AN Y e Background d i e Baseline e Experimental error such as contamination pipetting errors and so on can produce data that deviate Threshold significantly from data for typical amplification curves Such atypical data cause the software algorithm to generate incorrect baseline and threshold values for the associated detector Therefore Applied Biosystems recommends reviewing all baseline and threshold values after analysis of the study data If necessary adjust the values manually as described on page 69 Manual Baseline and Threshold Determination If you set the baseline and threshold values manually for any detector in the study you must perform the adjustment procedure on page 69 for each of the detectors The following amplification plots show the effects of baseline and thresho
85. ng Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System References 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 Livak K J and Schmittgen T D 2001 Analysis of Relative Gene Expression Data Using Real Time Quantitative PCR and the 2 T Method Methods 25 402 408 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 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 85 References 86 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Numerics 3D bars 74 5 nuclease assay 19 9600 Emulation Mode 37 A AIF See assay information files AmpErase UNG 20 amplification curve 66 amplification plots Amplification Plot view for RQ plates 39 57 types of amplification plots for RQ studies 72 appearance of graphs 38 56 73 74 Applied Biosystems contacting vili customer feedback on documentation 1x Services and Support viii Technical Communications 1x Technical Support viii assay information files 83 autoscaling options 38 56 73 74 B
86. nt applications such as allelic discrimination SNP Genotyping or Plus Minus Assays e Applied Biosystems has verified the performance of Applied Biosystems TaqMan Gene Expression Assays and Custom TaqMan Gene Expression Assays using the default Fast thermal cycling conditions and the TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG e It is expected that the vast majority of custom 5 nuclease quantification 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 Maser 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 59 e 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 59 for further information Notes 52 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Specifying Thermal Cycling Conditions and Starting the Run Default Thermal Cycling Conditions for PCR Default Thermal Cycling Conditions for PCR If you selected the two step RT PCR method for your RQ experiment re
87. on Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 5 Repeat steps 2 through 4 to set the baseline and threshold values for all remaining detectors in the study 6 Click or select Analysis gt Analyze to reanalyze the data using the adjusted baseline and threshold values Notes Adjusting the Baseline and Threshold Manual Baseline and Threshold Determination Delta Rn vs Cycle 5 H E 11 1a 15 Jy 1E 21 23 23 y 2m l a 33 ar z Cycle Number Click and drag the Threshold setting to adjust the threshold The bar turns red indicating that the threshold has been changed Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 71 SE Chapter 6 Analyzing Data in an RQ Study Analyzing and Viewing the Results of the RQ Study Analyzing and Viewing the Results of the RQ Study Selecting Detectors to Include in Results DS GB AlE b ones ose yee CAE fr S ton oaeo Graphs Find al Gene Expression SE In the RQ Detector Grid select detectors to include in o the result graphs by clicking a detector Ctrl click to a include multiple detectors Click drag to include qe multiple adjacent detectors The corresponding samples appear in the RQ Sample Grid Depending on which tab you select in the RQ Results Panel Plate Amplification
88. onditions 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 4343458 for more information Notes 60 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Chapter 6 Analyzing Data in an RQ Study Workflow Create a new RQ Study document See page 62 Configure analysis i See page 64 settings Adjust the baseline See page 66 and threshhold Analyze and view results See page 72 If necessary See page 77 omit samples Export AQ Plate i See page 79 document if desired Performing an RQ Study Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 61 Chapter 6 Analyzing Data in an RQ Study Creating an RQ Study Document
89. ow the handling instructions Wear appropriate protective eyewear clothing and gloves Nexen CHEMICAL HAZARD TaqMan Universal PCR Master Mix 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 Notes Reaction uL uL 5 pna Component Sample Reactions Sonor tration TaqMan Universal 25 0 125 0 1X PCR Master Mix 2X 20X TaqMan 2 5 12 5 1X Gene Expression Assay Mix cDNA sample 5 0 25 0 10 to 100 ng Nuclease free 17 5 87 5 water Total 50 0 250 a Contains forward and reverse primers and labeled probe b 24 master mixes are prepared one for each of 23 genes plus the endogenous control Volume for five reactions 4 replicates plus extra to account for pipetting losses PCR Master Mix Fast Reaction uL uL 5 Final Component Sample Reactions Concen tration TaqMan Fast 10 0 50 0 1X Universal PCR Master Mix 2X 20X TaqMan 1 0 5 0 1x Gene Expression Assay Mix CDNA sample 10 to 100 and 9 uL 45 uL ng Nuclease free water Total 20 0 100 8 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Example RQ Experiment Example RQ Experiment Procedure 5 Prepare the reaction plates
90. oward the front of the instrument 5 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 a message indicates whether or not the run is successful All data generated during the run are saved to the RQ Plate document that you specified in step 3 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 55 Analyzing and Viewing RQ Plate Data Chapter 5 Generating Data from RQ Plates 7500 Fast System Analyzing and Viewing RQ Plate Data Starting the Analysis To analyze RQ Plate data after the run click or select Analysis gt Analyze The SDS software mathematically transforms the raw fluorescence data to establish a comparative relationship between the spectral changes in the passive reference dye and those of the reporter dyes Based on that comparison the software generates four result views Plate Spectra Component and Amplification Plot About the Results Tab In the Results tab you can view the results of the run and change the parameters For example you can omit samples or manually set the baseline and threshold If you change any parameters you should reanalyze the data The Results tab has four secondary tabs each of which is described below Details are provided in the Online Help e To move betwe
91. plates into text files which can then be imported into spreadsheet applications such as Microsoft Excel 1 Select File gt Export then select the data type to GDM view Tools Instrument Analysis Window Help export Mew Ctrl M Open Chri oO e Sample Setup txt Close Save Chrl 5 e Calibration Data csv Save As xk Import Sample Setup e Background Spectra csv aE Component csv view Exported Results Calibration Data Page Setup Spectra e Rn csv Print Preview Component Print Ctrl P Rm Typically you export sample setup data for Te newly created and newly run plates other data 2 Kidney types are exported for existing plates Exit 2 Enter a file name for the export file Note The name of the dialog box depends on the type of data you want to export 3 Click Save Notes 58 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Troubleshooting Troubleshooting Troubleshooting Reanalyzing Data Observation 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 Quantif
92. products which provide predesigned primers and probes for the 23 genes 2 Isolate total RNA from liver kidney and bladder tissue as explained in Chapter 3 on page 24 3 Generate cDNA from total RNA using the High Capacity cDNA Archive Kit 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 Archive Kit Protocol Nee CHEMICAL HAZARD 10 x RT Buffer may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves b Prepare the cDNA archive plate by pipetting into each well of the plate e 50 uL RT master mix e 30 uL nuclease free water e 20 uL RNA sample Make sure the amount of total RNA converted to cDNA is 10 to 100 ng in 5 uL for each 50 uL PCR reaction Notes Example RQ Experiment Example RQ Experiment Procedure RT Master Mix Standard Plate Component uL Reaction as 5 a 10X Reverse 10 210 Transcription Buffer 25X dNTPs 4 84 10X random primers 10 210 MultiScribe Reverse 5 105 Transcriptase 50 U uL Nuclease free water 21 441 Total 50 1050 a Each RT reaction is 100 uL see step 3b If you need 5 uL cDNA for each of 104 PCR reactions per tissue see step 4 you need 6 RT reactions per tissue Extra volume enough for one additional RT reaction per tissue
93. quantities of a nucleic acid sequence If you want to perform relative quantification using the relative standard curve method you should use an AQ assay type and consult the Chemistry Guide for details on how to set up a run and analyze this type of assay Term Definition Baseline The initial cycles of PCR in which there is little change in fluorescence signal Threshold A level of AR automatically determined by the SDS 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 C7 The fractional cycle number at which the fluorescence passes the threshold 3 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Chapter 1 Introduction and Example RQ Experiment About RQ Experiments Term Definition 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 signal that is an indicator of spec
94. r accept the default 5 Click Next gt 6 Select detectors to add to the plate document a Click to select a detector Ctrl click to select multiple detectors If no detectors are listed in the Detector Manager create detectors as explained in Appendix A Creating Detectors b Click Add gt gt The detectors are added to the plate document Note To remove a detector from the Detectors in Document panel select the detector then click Remove Notes Chapter 4 Generating Data from RQ Plates 7300 or Standard 7500 System Creating a Relative Quantification RQ Plate Document Hew Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Relative Quantification ddCt Plate Y Container 96 W ell Clear E Template Blank Document ae Browse Operator Administrator Comments Default Plate Name Plate15 Finish Cancel New Document Wizard Select Detectors Select the detectors you will be using in the document Find i i FAM none GAPDH FAM none CD3D Remove CCr2 Detector Har none New Detector lt Back Next gt Finish Cancel 32 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Creating a Relative Quantification RQ Plate Document Creating an RQ
95. r TaqMan reagent based chemistry Multiplex PCR can use only TaqMan chemistry e Amount of time you want to spend optimizing and validating your experiment Amplifying target sequences and endogenous controls in separate reactions singleplex PCR requires less optimization and validation than multiplex PCR Among the factors to consider in multiplex PCR are primer limitation the relative abundance of the target and reference sequences the endogenous control must be more abundant than the targets and the number of targets in the study IMPORTANT As the number of gene targets increases the singleplex format is typically more effective than the multiplex format because less optimization is required Additionally running multiple reactions in the same tube increases throughput and reduces the effects of pipetting errors For more information about multiplex and singleplex PCR refer to the SDS Chemistry Guide PN 4361966 Example Experiment The singleplex PCR method is used in the example experiment because e The number of targets to be amplified 23 genes plus one endogenous control is large e Optimization and validation requirements are reduced for singleplex experiments Notes 16 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Specifying the Components of an RQ Experiment Sy A Specifying the Components of an RQ Experiment After
96. ression plots 74 omitting samples from 77 orientation 74 reanalyzing data 76 results 72 RQ Study documents 62 Index RT PCR one step 20 36 two step 20 35 53 S Services and Support obtaining viii settings graph 38 56 73 74 Setup tab 34 50 singleplex PCR 16 Spectra view 39 57 standard curves 2 standard deviation effect of threshold on 68 standard error 68 standard vs Fast plates 29 starting an RQ platerun 37 55 study RQ See RQ studies SYBR Green I dye chemistry 19 T TAMRA dye 39 57 82 TaqMan Assays 21 TaqMan Custom Gene Expression Assays 21 TaqMan MGB probes 22 82 TaqMan reagent chemistry 19 TaqMan Universal PCR Master Mix 28 TaqMan Gene Expression Assays 21 target associating with detectors 31 47 definition 17 tasks See detector tasks Technical Communications contacting 1x Technical Support contacting viii template documents 32 48 text conventions vii thermal cycling conditions default for PCR 35 53 one step RT PCR 36 specifying 36 54 two step RT PCR 35 53 thermal cycling parameters for plates added to a study 9 63 RT using High Capacity cDNA Archive kit 25 threshold adjusting 66 definition 3 examples 68 threshold cycle definition 3 setting for RQ studies 64 Training obtaining information about viii Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 89 Index U uracil N glycosylase 20 W
97. revention enzyme AmpErase UNG with one step RT PCR For more information about UNG refer to the SDS Chemistry Guide Notes Sample RNA RT Master Mix y RT Incubation Aliquot cDNA J Archive Standard PCR Master Mix OR Fast PCR Master Mix PCR PCR amplification amplification and detection and detection 1 hour 50 minutes lt 40 minutes One Step RT PCR Sample RNA RT Standard PCR Master Mix Master Mix y RT incubation and PCR amplification 2 5 hours 20 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Choosing the Probes and Primers Recommended Kits for Two Step RT PCR Chemistry Step Reagent Part Number TaqMan reagents or kits RT High Capacity cDNA Archive Kit 4322171 PCR TaqMan Universal PCR Master Mix 4304437 TaqMan Fast Universal PCR Master Mix 4352042 SYBR Green reagents or kits RT High Capacity cDNA Archive Kit 4322171 PCR SYBR Green Master Mix 4309155 RT and PCR SYBR Green RT PCR Reagents 4310179 Example Experiment Premade probes and primers for all the genes of interest are available from the TaqMan Gene Expression Assays product line which uses TaqMan reagent based chemistry Two step RT PCR is performed using the reagents recommended for TaqMan reagent or kit based chemistry in the table above Choosing the Probes and Primers
98. s era ee aed eas r E te tate tata ding bao A ts ate nee ey 41 Before YOU BeOIN sare Kiet eA eas heg ad Koti ee oe eae eek eR eae eee 42 Preparing the PCR Master Mix 0 000 ee eee eee eee 42 Preparing the Reaction Plate 0 ce eee 44 Creating a Relative Quantification RQ Plate Document 0020 05a 47 Specifying Thermal Cycling Conditions and Starting the Run 52 Analyzing and Viewing RQ Plate Data 0 0 0 ee 56 Exponing RO Plate Data sister neari aE e beoe et awin bees cea ewan bs 58 IMOUDIEGSHOOHNG asics othe tule eee ee ved eee ee te Ode wide aah ae 59 Chapter6 Analyzing Data in an RQ Study 61 NV ORK TOW testes sou dept eis tag ay toe Seater we oe ees A at aha eee ee a tee 61 Creating an RQ Study Document 2 00 cc eee ees 62 Configuring Analysis Settings 2 0 00 cc eee eens 64 Adjusting the Baseline and Threshold 0 00 0 eee 66 Analyzing and Viewing the Results of the RQ Study 0000 c eee eee 72 Reanalyzing an RQ Study sodenn apa a a a eee eae 76 Omitting Samples from a Study 0 00 ee eens 77 Exporting RQ Study Data lt cvewi ccs eh ee ae we bh eth Se a alee Soe awd 79 Appendix A Creating Detectors 81 References 85 Index 87 vi Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Preface How to Use This Guide Purpose of This This manual is written for principal
99. ses Printed in the USA 12 2004 Part Number 4347824 Rev C an Applera business
100. stems Chemistry Guide SDS Chemistry Guide and the Online Help for the 7300 7500 7500 Fast system Online Help About Relative Quantification Definition Real time PCR Assays Notes Relative quantification determines the change in expression of a nucleic acid sequence target in a test sample relative to the same sequence in a calibrator sample The calibrator sample can be an untreated control or a sample at time zero in a time course study Livak and Schmittgen 2001 For example relative quantification is commonly used to compare expression levels of wild type with mutated alleles or the expression levels of a gene in different tissues RQ provides accurate comparison between the initial level of template in each sample without requiring the exact copy number of the template Further the relative levels of templates in samples can be determined without the use of standard curves RQ is performed using real time PCR In real time PCR assays 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 There are two types of quantitative real time PCR absolute and relative 2 Relative Quantification Getting Started Guide for the Appli
101. 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 Fast Universal PCR Master Mix 10 0 1X 2X Notes 42 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Preparing the PCR Master Mix Fast System Requirements Reaction Component uL Sample Final Concentration Forward primer 2 0 50 to 900 nM Reverse primer 2 0 50 to 900 nM TaqMan probe 2 0 50 to 250 nM cDNA sample 2 0 10 to 100 ng Nuclease free water 20 Total 20 0 Probes and primers you design using Primer Express software must be optimized to work with the universal assay conditions using the volumes listed in the table above All TaqMan Custom Gene Expression Assays and TaqMan Gene Expression Assays are formulated so that the final concentration of the primers and probes are within the recommended values Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 43 Chapter 5 Generating Data from RQ Plates 7500 Fast System Preparing the Reaction Plate Preparing the Reaction Plate Fast vs Standard Plates IMPORTANT Make sure that you use the Optical 96 Well Fast Plate for fast gene quantification Standard plates will not
102. t you view the Amplification and Gene Expression plots If you then want to use Kidney or Bladder as the calibrator you need to reanalyze the data before viewing results Pa l Ki Similarly if you want to change the baseline or ny threshold values the endogenous control the control type or the RQ Min Max parameters you need to reanalyze your data Bladder o E amp g a oOo Notes 76 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Omitting Samples from a Study Omitting Samples from a Study Experimental error may cause some wells to amplify insufficiently or not at all These wells typically produce C values that differ significantly from the average for the associated replicate wells If included in the calculations these outlying wells outliers can result in erroneous measurements To ensure precise relative quantification you must carefully view replicate groups for outliers You can remove outliers manually using the C vs Well Position Amplification Plot To remove samples from an RQ Study 1 Select the Amplification Plot tab 2 Inthe Data drop down list select Ct vs Well Position 3 In the RQ Detector grid select a detector to examine All samples that use this detector are displayed in the RQ Samples grid Bladder B10 Bladder CCR2 374 4 Inthe RQ Samp
103. t the run tart the run PCR master mix reaction plate RQ Plate document Start the ru Start the ru Generating Data from Chapter 5 RQ plates Fast Performing Create a new Configure analysis Adjust the baseline Analyze and If necessary Export AQ Plate an RQ Study RQ Study document settings and threshhold view results omit samples document if desired Te Chapter 6 IV Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Contents RQ Experiment Workflow iil Preface vil HOW tO USE This GUIE 24th ee ke aie hae Re Uae ae eo ae aed nee eee eS vil How to Obtain More Information anana ccc eee eee Vill How to Obtain Services and Support 0 0 0 ce ee eee viii oend US YOur COMMENIS cin ees ea eat aad Pee eee Sears eS e Ix Chapter 1 Introduction and Example RQ Experiment 1 OVENI 2d ci alist hat cakes ck Seta usc aad Bn act sae ok hela ccna ay dah a dee ale ae a ace i aha ad aE 1 About the 7300 7500 7500 Fast System 0 cc eee 2 About Relative Quantification 0 0 ees 2 ADCULIR EXDeimeGntS a te 6c5 4 sth cade hae ad echt eh Gam Baca ke dated ace dt stun tee 2 3 Example RQ EXpeniMeni i lt iuiecaacada cwatd ebaven ound ehawe bri 6 Chapter2 Designing an RQ Experiment 15 WOKO manae Sa E caveats pune ata de E ide eae ne Sie see ae at ngs Men Armia
104. ted on the x axis and each bar shows the set of sample values of a single detector n o o _ Cc Ci oa amp a He D a Ea Kidney Liver Error Bars for Gene Expression Plots The SDS software displays error bars for each column in the plot provided that the associated expression level was calculated from a group of two or more replicates The error bars display the calculated maximum RQMax and minimum RQMin expression levels that represent standard error of the mean expression level RQ value Collectively the upper and lower limits define the region of expression within which the true expression level value is likely to occur The SDS software calculates the error bars based on the RQMin Max Confidence Level in the Analysis Settings dialog box see page 64 Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 75 SSS Chapter 6 Analyzing Data in an RQ Study Reanalyzing an RQ Study Reanalyzing an RQ Study If you change any of the analysis settings you must reanalyze the data before you can view results You can switch between the variations of the Liver Amplification and Gene Expression plots without having to reanalyze the data Calibrator Gene Expression Plot Suppose you select Liver as the calibrator then perform an analysis Nex
105. uantification ddCt Study Quantification ddCt Study Accept the default OO settings for Container and Template ice ooo plate 96 Well Clear and Blank Document Operator Administrator 3 Enter a name in the Default Plate Name field or Oo accept the default omments p Default 4 Plate Name Platet 5 Click Next gt Finish Cancel Notes 62 Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Creating an RQ Study Document 5 Add RQ plates to the study a Click Add Plates Heave Dun uml Wicca A dd Plate Sud lates to the FQ hiey Select RO Plite a Note You can add up to 10 RQ plates to an RQ study Look n FI rletes fe F E 7 F 7 E FEE Mu Fel E il Dera imal b Select the plate s that you want to add to 1 sk p the study then click Open W biy Compule Ei 4 Plate Z si Naso 1A DAME Tadarida dep sda Dower ac Opon Add Plates Ooo E omn The selected plates are displayed _ Seemann merase x e eeeoeeeeo O S sSmm930 0 0 I I Add Plates IMPORTANT All plates added to a study AAA P E etl must have identical thermal cycling n parameters the same number of steps T PEET cycles sample volume emulation mode Kidney Aug 05 2003 02 05 PM Liver Aug 05 2003 02 05 PM The
106. when you set up the plate document Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 59 Chapter 5 Generating Data from RQ Plates 7500 Fast System Troubleshooting Observation Possible Cause Action 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 variability 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 Use reagents that contain ROX passive reference dye 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 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 c
107. xperiment is singleplex there is only one sample either a target or endogenous control in each well Each well is associated with a detector indicated by the colored squares Additionally each well is assigned a detector task T target or E endogenous control The figure below shows the example RQ Plate document after sample names detectors and detector tasks are assigned for each well in the liver plate Sample name D Uver Leer Uvar LBre Uvar Leer Uvar Lhe Uvar Leer Uvar Leer Liver E Uver Lhe Liver ie ier liwe ier iwe lr lier 1 Detector task and H a T ia H E color a Uvar Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre H E m E m H H i Uvar Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre H H H H H i Uvar Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre Uvar Lre EI E fal a fal LE El EJ Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 51 Chapter 5 Generating Data from RQ Plates 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 Run assays using Fast thermal cycling conditions e Applied Biosystems has verified the performance of Fast thermal cycling and the TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG for quantitative applications only and not for endpoi
108. y the amount of cDNA template 2 Test the cDNA template for the presence of PCR inhibitors Sample degradation Prepare fresh cDNA then repeat the experiment The TaqMan 2X Universal PCR Master Mix was used instead of the TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG Prepare the reactions with the correct Master Mix 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 Low AR or R values Extension time is too short Use the default thermal profile settings See page 53 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 Run takes more than 40 minutes Thermal cycler mode is set to Standard or 9600 Emulation Make sure that the thermal cycler mode is set to Fast See page 54 Rn vs Cycle plot is not displayed ROX dye was not selected as the passive reference when the plate document was set up Select ROX dye as the passive reference
109. you decide to use the singleplex or multiplex method specify the required components of the RQ experiment for every sample e A target The nucleic acid sequence that you are studying e A calibrator The sample used as the basis for comparative results e An endogenous control A gene present at a consistent expression level in all experimental samples By using an endogenous control as an active reference you can normalize quantification of a cDNA target for differences in the amount of cDNA added to each reaction Note that Each sample type for example each tissue in a study comparing multiple tissues requires an endogenous control Ifsamples are spread across multiple plates each plate must have an endogenous control Additionally every plate must include an endogenous control for every sample type on the plate Typically housekeeping genes such as B actin glyceraldehyde 3 phosphate GAPDH and ribosomal RNA rRNA are used as endogenous controls because their expression levels tend to be relatively stable Replicate wells For relative quantification studies Applied Biosystems recommends the use of three or more replicate reactions per sample and endogenous control to ensure statistical significance For more information about these requirements refer to the SDS Chemistry Guide Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR Syste
110. ys the e Associated samples from all plates included in the study in the RQ Sample grid e Graph for the selected detector in the RQ Results panel Note When manually adjusting baseline and threshold settings you can select only one detector at a time If you select multiple detectors the Analysis Settings section and the threshold bar are disabled Notes Relative Quantification Getting Started Guide for the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System 69 SSS Chapter 6 Analyzing Data in an RQ Study Adjusting the Baseline and Threshold 3 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 is enabled indicating that you must reanalyze the data 4 Set the threshold for the detector a Under Analysis Settings select Manual Ct b Drag the threshold setting bar so the threshold is e Above the background e Below the plateaued and linear regions of the amplification curve e Within the exponential phase of the amplification curve The SDS software adjusts the theshold value and displays it in the Threshold field after reanalyzing Notes 70 Relative Quantificati

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