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Relative quantification guide

1. 30 sis hun geh 4 amp idney 25 5 Verify the uniformity of each replicate d E 0 l l Plate Y Amplification Plot Y Gene Expression X population by comparing the groupings of Cy ce values for the wells that make up the set or Paso 2 pee e Good Fra 0320520 Aut clustering aa ie of replicate em ssn us p data No outliers Em PUES RE j Potentia PTRA E i i outlier a Be Notes 50 Absolute Quantification Getting Started Guide for the 7300 7500 System 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 f Repeat steps 5 and 6 to screen the remaining replicate groups 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 screen Notes Omitting Samples from a Study Bladder B10 Bladder CCR2 Well Information Select Omit Bladder B10 Bladder CCR2 37 592 0 305 w The outlier is removed during analysis Absolute Quantification Getting Started Guide for the 7300 7500 System 51
2. 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 26 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 of the 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 eeoeoec0 600000 eeoeococ c6co0o00000 eeoeoc00e606000200 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 21 The cDNA is stored at 20 C until use Notes 22 Absolute Quantification Getting Started Guide for the 7300 7500 System Chapter 4 Workflow Notes Prepare the See page 24 PCR Master Mix
3. RT Master Mix Component uL Reaction i i im 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 is included to account for pipetting losses as well as extra cDNA for archiving Liver Kidney Bladder a e a 2 EE EEEE EEEE ED EEEE E 0000o eeoooe0 eeoooe0 eeooo e eeoooe eooo2o9 9 eooeooeo9 eeoooe0 e ee0e 0 eee0e 0 eee0e 80 6 Relative Quantification Getting Started Guide for the 7300 7500 System c Program the thermal cycler using the indicated parameter values for the RT step of the two step RT PCR method Note You have the option to use one step RT PCR as explained in Selecting One or Two Step RT PCR on page 16 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 for more information Note The reaction volumes for Assay by Design products are specified in the product insert those for primers and probes designed with Primer Express software follow the universal assay conditions de
4. Chapter 5 Analyzing Data in an RQ Study 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 l View Tools Instrument Analysis Window Help New Ctrl M data type to export Hd BUE e Sample Summary csv Eis p y Save Ctri 5 Well Information csv di Import Sample Setup Both csv Export Sample Setup View Exported Results Calibration Data Refer to the Online Help for information about sare cet age Setup pectra the export file types Print Preview Component Print Ctrl F Delta Rn Ch 1 RNAse P 2 14 05 2 ROStudy1 Results Li Sample Summary 3 Liver Well Information 4 Liver_InProgress Both 5 RaPlateTesti 6 E cali reading 7 E coli Plus Minus Pre Read 8 E cali amplification QE coli readingzinstruTab Exil 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 52 Absolute Quantification Getting Started Guide for the 7300 7500 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 allel
5. Create a new See page 26 RQ Plate document Create detectors See page 26 Program the See page 30 thermal cycling conditions Save the See page 31 RQ Plate document Generating Data from RQ Plates Start the run See page 31 View RQ plate data See page 32 Absolute Quantification Getting Started Guide for the 7300 7500 System 23 Chapter 4 Generating Data from RQ Plates 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 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 The TaqMan Universal PCR Master Mix Protocol PN 4304449 explains how to use the reagents in the kit The following table lists the universal assay conditions volume and final concentration for using the master mix N eui 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 TagMan Universal PCR Master Mix 2X 25 0 1X Forward prime
6. 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 graph Fin a Plate Y Amplification Plot Y Gene Expression 0 20 Log 10 Relative Quantification 1 20 1 40 jee 6QR 28 lt gt Sample Summary Well Information E Omit Date Delta Rn vs Cycle v Calibrator iver Orientation Detector E Cc o o o 3 G o E amp a a x a e c o d vR1 VR R2 CD3D FLT4 GAPDH Detectors je ae E fe Omit Data Delta Rn vs Cycle Calibrator Liver Orientation 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 e Because t
7. 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 TaqgMan 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 8 s 7 se s J Liver 5 34 Liver T 552 Liver T 4 58 Liver 7 01 Liver T 548 Liver 5 31 Liver T 528 Liver IT 4 84 Liver 6 94 Liver T 545 Liver 5 23 Liver T 6 36 Liver m 470 Liver 6 97 Liver T540 Raw Data Liver fa 5 10 Liver T 572 Liver m 476 Liver 7 00 Liver T 514 Liver 7 83 Liver m 246 Liver T 6 38 Liver B 85 Liver IT ERE Liver Bs Liver ds Liver T8548 Liver Hes Liver H i Setup Instrument Y Results Plate Spectra Y Component Amplification Plot Component mm SEO EGG Gahan UL Lean ee 3200 00 2800 00 2400 00 2000 00 1600 00 Fluorescence 1200 00 800 00 400 00 Setup Instrument Results Plate Y Spectra Y Component Y Amplification Plot Cycles Rn vs Cycle 9g 11 13 15 36 19
8. 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 40 To configure analysis settings Click m or select Analysis gt Analysis Settings 2 In the 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 and Threshold on page 40 Alternatively you can select Manual Ct and specify the threshold and baseline manually Notes Analysis Settings Relative Quantification Ct Analysis Detector All n Auto Ct manua Ot 3 Threshold 0 200000 5 Start cycle Auto End cycle Auta Gene Expression Calibrator 5 ample E Endogenous Control Detector GAPDH ES Control Type Mon multiplexed ey RO Min Max Confidence 35 UU Replicate and Outlier Removal Iv Remove Outlier OK amp Reanalyze Cancel Apply 38 Absolute Quantifi
9. 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 Absolute Quantification Getting Started Guide for the 7300 7500 System 13 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 FLTA 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 fr
10. 3D bars 47 5 nuclease assay 15 9600 Emulation Mode 31 A AIF See assay information files AmpErase UNG 16 amplification curve 40 amplification plots Amplification Plot view for RQ plates 33 types of amplification plots for RQ studies 45 appearance of graphs 32 46 47 Applied Biosystems contacting vili customer feedback on documentation viii Services and Support viii Technical Communications viii Technical Support viii assay information files 54 Assays by Design 17 Assays on Demand 17 autoscaling options 32 46 47 B background in amplification curve 40 bar width 47 baseline adjusting 40 defintion 3 examples 41 C calibrating the 7300 7500 instrument 24 calibrator definition 13 selecting in RQ studies 39 CAUTION description vii cDNA generating 21 storing 22 See also reverse transcription Index Chemistries 15 comparative method of calculation 3 36 Component view 33 concentration of RNA 20 confidence levels 39 conventions text vii Ct See threshold cycle curves amplification 40 standard 2 D data analyzing 32 45 exporting 34 52 generating PCR data from RQ plates 31 importing 27 omitting from a study 50 deltaRn 3 Delta Rn vs Cycle view 46 designing RQ experiments determining reagent configuration 15 PCR method 12 primers and probes 17 selecting the chemistry 15 Detector Manager dialog box 53 detector tasks definition 26 detectors adding to RQ plates 27 creati
11. AmpErase UNG 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 Sample RNA RT Master Mix i y RT Incubation Aliquot DNA J Archive PCR Master Mix PCR Amplification and Detection Results One Step RT PCR 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 prevention enzyme AmpErase UNG uracil N glycosylase with one step RT PCR For more information about UNG refer to the SDS Chemistry Guide Sample RNA RT PCR Master Mix Master Mix y RT Incubation and PCR Amplification A 4 F Results Notes 16 Absolute Quantification Getting Started Guide for the 7300 7500 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 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 in
12. 0 0 ce eee eee eens 38 Adjusting the Baseline and Threshold 0 00 eee 40 Analyzing and Viewing the Results of the RQ Study 0 002000 ce eee 45 Reanalyzing an BO Study 4e Bee eee Se hee ele ARIA KE SES abs 49 Omitting Samples from a Study 0 0c a 50 EXDOminG RO Study Data tuo he i ee roe ar cance Sat eo de doe a Qe hah ate aan 52 Appendix A Creating Detectors o3 References 99 Index 57 vi Relative Quantification Getting Started Guide for the 7300 7500 System Preface How to Use This Guide Purpose of This This manual is written for principal investigators and laboratory staff who conduct Guide relative quantification studies for gene expression using the Applied Biosystems 7300 7500 Real Time PCR System 7300 7500 system Assumptions This guide assumes that you have Familiarity with Microsoft Windows XP operating system Knowledge of general techniques for handling DNA and RNA samples and preparing them for PCR A general understanding of hard drives and data storage file transfers and copying and pasting If you want to integrate the 7300 7500 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 talic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matr
13. 12 singleplex 12 starting an RQ plate run 31 phases of amplification curve 40 Plate view 33 plate RQ See RQ plates plateau phase of amplification curve 40 plot appearance 32 46 47 Primer Express Software 17 primers 17 probes 17 54 H reagent configurations 16 real time PCR assays 2 reference sample See calibrator reference passive 3 28 relative quantification comparative method of calculation 3 definition 2 example experiment 5 experiments See also RQ experiments 2 real time PCR 2 references 2 RQ plates 3 RQ studies 3 replicates 13 reporter dye 3 Absolute Quantification Getting Started Guide for the 7300 7500 System Results tab 32 results RQ Study analysis 45 reverse transcription guidelines for preparing RNA 20 High Capacity cDNA Archive kit 21 thermal cycling parameters 21 Rn vs Cycle view 46 Rn See normalized reporter RNA guidelines for preparing 20 isolation 20 starting concentration 20 ROX dye 28 33 RQ Detector grid 39 RQ experiments chemistries 15 components 13 probes and primers 17 reagent configuration 15 requirements 13 workflow 2 RQ Main Study view 37 RQ plates adding to RQ studies 37 Amplification Plot view 33 analyzing 32 Component view 33 datatypes 34 definition 3 detectors creating 53 exporting data 34 Plate view 33 reanalyzing data 34 results 32 RQ Plate documents 26 Spectra view 33 staring a run 31 RQ Results panel 45 RQ Sample grid 45 RQ studies adding
14. 21 23 25 727 29 31 34 35 31 39 Cycle Number Component s Mv Fam Hv Rox Detector Aq Line Color well Color gt Absolute Quantification Getting Started Guide for the 7300 7500 System 33 Chapter 4 Generating Data from RQ Plates 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 i 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 EE view Tools Instrument Analysis Window Help export Mew Ctrl Open Chri a e Sample Setup txt Close Save Cris Calibration Data csv Save As xk Import Sample Setup Background Spectra csv HN e Component csv View Exported Results Calibration Data Page Setup Spectra e Rn csv Print Preview Component Print Ctrl P Rn Typically you export sample setup data for T newly created and newly run plates other data 2 Kidney types are ex
15. RNA from Whole Blood and from Cells Isolated from 4332809 Whole 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 Quality of RNA The total RNA you use for RQ experiments should e Have an Aoe95s9 greater than 1 9 Beintact 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 20 Absolute Quantification Getting Started Guide for the 7300 7500 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 IMPORTANT The protocol is not shipped with the High Capacity cDNA Archive Kit Download the protocol from http docs appliedbios
16. RQ plates 37 Amplification Plots 45 confidence level 39 control type 39 data types 52 definition 3 exporting data 52 Gene Expression plots 47 omitting samples from 50 orientation 47 reanalyzing data 49 results 45 RQ Study documents 36 Index RT PCR one step 16 30 two step 16 30 S Services and Support obtaining viii settings graph 32 46 47 Setup tab 29 singleplex PCR 12 Spectra view 33 standard curves 2 standard deviation effect of threshold on 42 starting an RQ plate run 31 study RQ See RQ studies SYBR Green I dye chemistry 15 T TAMRA dye 33 54 TaqMan chemistry 15 TaqMan MGB probes 18 54 TaqMan Universal PCR Master Mix 24 target associating with detectors 26 definition 13 tasks See detector tasks Technical Communications contacting viii Technical Support contacting viii template documents 27 text conventions vii thermal cycling conditions default forPCR 30 one step RT PCR 30 specifying 31 two step RT PCR 30 thermal cycling parameters for plates added to a study 37 RT using High Capacity cDNA Archive kit 21 threshold adjusting 40 definition 3 examples 42 threshold cycle definition 3 setting for RQ studies 38 Training obtaining information about viii U uracil N glycosylase 16 Absolute Quantification Getting Started Guide for the 7300 7500 System 59 Index W WARNING description vii Well Information tab 45 wells replicate 13 width bars 47 w
17. experiment Select one step or two step RI PCR Save the RQ Plate document RQ Experiment Workflow Choose probes and primers Start the run View RQ plate data Performing Create a new Configure Adjust the Analyze and Chapter 5 an RQ Study RQ Study document analysis settings re view results resho Relative Quantification Getting Started Guide for the 7300 7500 System If necessary omit samples Export AQ Plate document if desired Relative Quantification Getting Started Guide for the 7300 7500 System Chapter 1 Chapter 2 Chapter 3 Chapter 4 Contents RQ Experiment Workflow iii Preface vil How1o Use This GUIGO e op E ko eee OR Bob ode RC Rate coe wae Ree ees vii How to Obtain More Information lcs Viii How to Obtain Services and Support 0 0 0 0 ce ee eee viii Send US Your C OTtie ls Vatt foie dana o Scu ded Mira vidct ied Roter pw a viii Introduction and Example RQ Experiment 1 9l rM EE 1 About the 7300 7500 System 43 3 cx m am Ro dE E EI Ra ER REI EX RC o ee Col ca 2 About Relative Quantification lllleeeeeeee rrr 2 About fao EXDeFHITIGPBIS s cies ui cabe die dp Rc CE e ep dep rbd b e e d oe Ee c d 2 Example RO EXpenimenis lt ictocencacd doc 3 MORARI En EO Pb d qo ad C PUE fc qd did 5 Designing an RQ Experiment 11 tios ajo FX rM PENES 11 Selecting the PGR Metod 43 3c noci S EA o
18. related tyrosine kinase 4 Hs00176607 m1 GAPDH glyceraldehyde 3 phosphate dehydrogenase Hs99999905 m1 Notes 18 Absolute Quantification Getting Started Guide for the 7300 7500 System Primer Extended on mRNA 5 Performing Reverse Transcription Oligo Synthesis of 1st cDNA strand F Workflow Performing Reverse Transcription Isolate total RNA See page 20 Adjust RNA See page 20 concentration pag Convert total RNA to cDNA See page 21 Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 19 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 System 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 analysis using the 6100 PrepStation Isolation of Total
19. 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 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 specific 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 Relative Quantification Getting Started Guide for the 7300 7500 System 3 Chapter 1 Introduction and Example RQ Experiment About RQ Experiments The figure below shows a representative amplification plot and includes some of the terms defined in the previous table Threshold No Template Control 0 5 10 15 20 25 30 3
20. 000 09 0 0 0 0 0 0 0 O O O Samples eoeoooooooooooooo020200 00000000000 GISISTO GISISTO GIVISIO Endogenous 000000000000 e eeeeeeeOddod 000000000080 controls GAPDH 9999 99999 9 9 9 Endogenous controls GAPDH eooooeoooooo00200 eeoeooooooooooo202090 eoeoooooooooooo00200 Kidne eececcc606060000 iei ecece06006000000 o 0o 0 O0 O O O O O O O O GISISTO GISISTO 0 980 6 6 6 06 0080 O O O T controls GAPDH eeoeooooooooooooo0o0 0e eoooooooooooooo000 0 eeoeooooooooeooooo0P9 Bladder 0 0 0 0 0 0 0 0 08 00 npes eoeooooooooooo 0090 eeooeoooooooooPe020 009 0 o 0 O O O O O O O O O O am DD Aaa DA aD a A Endogenous Notes 14 Absolute Quantification Getting Started Guide for the 7300 7500 System Selecting the Chemistry About Selecting the Chemistry About Chemistries 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 Description TaqMan reagent based chemistry uses a fluorogenic probe to enable detection of a specific PCR product as it accumulates during PCR cycles Advantages e Increases specificity w
21. 5 4 Cycle Number Required User Supplied Item Source Materials f ABI prism 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 MicroAmp Optical 96 Well Reaction Plate Applied Biosystems PN 4306757 Optical Adhesive Cover Applied Biosystems PN 4311971 Labeled primers and probes from one of the following sources e Assays on Demand Gene Expression e Applied Biosystems Web site Products predesigned primers and probes e Assays by Design service predesigned e Contact your Applied Biosystems Sales primers and probes Representative e Primer Express Software custom e PN 4330710 1 user license designed primers and probes PN 4330709 10 user license PN 4330708 50 user license 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 Notes 4 Relative Quantification Getting Started Guide for the 7300 7500 System Example RQ Experiment Overview Item Source Nuclease free water MLS Pipette tips with filter plugs MLS Pipettors positive displacement MLS Safety goggles MLS Vortexer MLS Example RQ Experiment
22. 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 c Click Next gt Notes Creating an 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 Container 35 w ell Clear m Template Blank D acument E Brawse Operator Administrator Comments Default Plate Mame Plate15 Finish Cancel New Document Wizard Select Detectors Select the detectors you will be using in the document Find a 1e Quenche Detectors in Document FAM none GAPDH FAM none FLT4 Add gt gt ree Remove Clr A Detector Har none New Detector lt Back Next gt Finish Cancel Absolute Quantification Getting Started Guide for the 7300 7500 System 2 Chapter 4 Generating Data from RQ Plates Creating a Relative Quantification RQ Plate Document f Specify the detectors and tasks for each well n
23. Inc 1145 Atlantic Avenue Alameda California 94501 USA Trademarks Applied Biosystems MicroAmp Primer Express ROX and VIC are registered trademarks of Applera Corporation or its subsidiaries in the US and or certain other countries AB Design ABI PRISM Applera Assays by Design Assays on Demand Celera Genomics FAM 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 A 1 2004 Relative Quantification Getting Started Guide for the 7300 7500 System Introduction Chapter 1 and Example About the About relative About RQ Experiment 7300 7500 system quantification RQ experiments Select the Specify the components Chapter 2 i PCR method of an RQ experiment Performing Chapter 3 aaan a Reverse Isolate total RNA 7 Transcription Adjust RNA Convert concentration total RNA to cDNA Prepare the Prepare the Create a new Program the Chapter 4 Muf Data from PCR Master Mix reaction plate RQ Plate document thermal cycling RQ Plates conditions Example RQ
24. Li Ectup Samplc Plate a Click a well or group of wells for Setup che sampl pare ath tases quant tiec and dztectzre uea Detecrar Reparar xencher laak Color replicates to select it aH ak inne Tae b Click to select the detector s for the well Mw ACVvREI FAM none Target E Lr T 0 0 8 LEN ETE n ne Fat ENDO s rf e wfliw 2 RPVRT c Click under the Task column to assign the detector task d Select Use e Click Finish H E d La M e 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 well s and type text directly into the sample name SDS v1 2 Software Note You will need to enter sample names before saving the RO Plate document The SDS software creates the plate After editing sample name s vou can then save the RO Plate document 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 Pe LL Liver Liver b Enter the sample name c If necessary change the setting for the Passive Reference dye By default the ROX dye is selected d Repeat steps a through c until you specify sample names and passive re
25. Notes Overview Description 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 The objective of the example RQ 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 Assays on Demand 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 th
26. OOOO OOOO EO amples ececc00000000 ececc006000000 9 0 0 0 0 0 0 0 8 9 8 O E dogenous 06 0 00 000 6 oO O OF controls GAPDH ececcc060060000 eecc060000000 eec0000000099 90 090 0 0 80 SSIS 7 0000000008008 i ececc060000000 9 09 00 0 9 8 0 0 990 0 0 0 0 9 0 0 0 O O OF controls GAPDH eeccccc60606000 ecececcccccccoo 00000000008 Qo ice 0006600000009 samples eeccc0c6000000 ecececccccccoco 9 0 0 0 0 0 0 0 0 89 88 o O O O O O O O O D O O OF controls GAPDH Relative Quantification Getting Started Guide for the 7300 7500 System Chapter 1 Introduction and Example RQ Experiment Example RQ Experiment 6 Create an RQ Plate document as described in Hew Document Wizard Creating a Relative Quantification RQ Plate Define Document Document on page 26 Briefly Rd EA container and template for the document and enter the operator name and a Select File gt New Assay Relative Quantification ddCt Plate b Select Relative Quantification ddCt oo Wel Clea T Plate in the Assay drop down list then B Plank Document 7 click Next gt Browse E M Operator Administrator 00 IMPORTANT You cannot use AQ Plate T documents for RQ assays and vice versa EE The information stored in AQ and RQ Plate n documents is not interchangeable PhieNamePBel8 00000 c Add detectors to the plate document then Finish Cance
27. 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 To move between views click a tab To select all 96 wells on a plate click the upper left corner of the plate To adjust graph settings 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 E File view Tools Instrument Analysis Window Help Du E amp El EF ES Setup Y instrument Y Results V Plate Spectra Y Component Y Amplification Flot setup Y instrument Yresutts V Plate Spectra Y Component Y Amplification Flot Ds s 5 Bd i Liver Liver IB 5 34 5 31 Liver Liver Liver Liver Liver Liver 5r 5 93 5 95 5 97 5 52 5 28 Graph Settings Real Time Setti
28. Relative Quantification oco Applied Biosystems 7300 7500 Real Time PCR System RQ Experiment Designing an RQ Experiment Performing Reverse Transcription A O Applied O m AS Biosystems Generating Data from RQ Plates 7500 Real Time PCR 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 Serial No 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 pr
29. You can use this plot to locate outliers from detector data sets see Omitting Samples from a Study on page 50 for more information Notes r Omit Data Rin vs Cycle Calibrator Liver B Plate Amplification Plot Y Gene Expression Y 1 3 5 7 9 11 13 145 17 19 21 23 25 27 29 31 33 35 37 39 Cycle Number 9 Omit Date RETINENS Calibrator Liver Orientati Plate Y Amplification Plot Y Gene Expression Y Delta Rn vs Cycle 1 0e 001 1 0e 000 1 0e 001 Delta Rn 1 0e 002 Mes 1 0e 003 1 0e 004 1 3 5 7 9 14 13 15 17 19 21 23 25 27 29 31 33 35 37 39 Cycle Number E Omit Data Ctvs Well Position Calibrator Liver Orie Plate Y Amplification Plot Y Gene Expression V Ct vs Vell Position Ct D 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 B1 65 69 73 77 81 85 89 93 Well Position 46 Absolute Quantification Getting Started Guide for the 7300 7500 System 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 You can adjust graph settings for gene expression plots in the Graph Settings dialog box including Bar width e 3D bars Autoscaling e Data display as Log RQ or Raw RQ Refer to the Online Help for more information about ad
30. a CD3D CD3D Liver Target 33 449 12 327 0 114 Target 34 493 14 567 0 449 Target 38 314 16 344 0 377 Kidney Bladder CO3D Notes Dalta Rn Delta Rn Delta Rin a eae ee Er eee oped te bac tc av hs eR ES acq Cycle Number 1 35 7 8 11 13 15 17 1H 21 23 25 27 2U 31 33 32 37 39 Cycle Number Move down g ios uU EEEE Beed IS BERI B PAg ET EEA BT A8 EI meat Eee ZUESTI X333 Cycle Number 42 Absolute Quantification Getting Started Guide for the 7300 7500 System Adjusting the Baseline and Threshold Manual Baseline and Threshold Determination a jue d sl Es E Omit Data Delta Rive ce E Calibrator Liver Orientation Dete EEE IEA EREEREER SEE EE RR ERSS r 5 T E End 000000 ban a Plate Y Amplification Plot Gene Expression Y Delta Fn vs Cycle 0 535276 Auto 1 0e 001 ACYR2 O353770 Auto Auto O 056001 Auto 0 267214 Geometric 1 0e 000 phase of the 0 200000 amplification FLT4 0451420 Auto Auto 1 0e 001 curve GAPDH 0 418936 Auto Auto c GTF2B 0 251278 Auto Auto us GTFal 0 280024 Auto Auto a Bo 4 e002 p HTR4 0082586 Auto Auta 1 0e 003 Sample Detector Task 1 0e 004 Liver CD3D Target 274376139 01421 0 000 1 000 135 7 811131517 19 21 23 25 2r 28 3 33 35 37 38 Kidney CD30 Target 268 158 68190 0194 2 051 0 241 Bladder CD30 Target 31 519 10 007 0 172 wrle Miimhnar ap Analysis Settings A
31. cation Getting Started Guide for the 7300 7500 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 f Select the RQ Min Max Confidence level 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 48 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 50 9 Click OK amp Reanalyze The detector V Amplification Plot Y Gene Expression Y information appears in the RQ Detector grid Detector Threshold AutoCT Baseline s EE Moaea ACYR1 0 535276 Auto Auto Kidney a 05 2003 02 After analysis th
32. d eo ER ce b b RE REIHE RE 12 Specifying the Components of an RQ Experiment 2 0 00 cee eee eee 13 Selecting the Chemistry 000 cece ee eee rn 15 Selecting One or Two Step RT PCR 2 0 eens 16 Choosing the Probes and Primers eee eee eens 17 Performing Reverse Transcription 19 MVOEKIIQNV i acter Ren uda eae a d EV xau ER Ada E SRI ERES S 19 Guidelines for Preparing RNA 00 ce rn 20 Converting Total RNA to CDNA 0 00 cc eens 21 Generating Data from RQ Plates 23 NVOMITIOW ux oia Bossi dc bos DOC o UR ODE AE CS aS oe Gane ta Odo CO NC cma MR 23 Belore YOU BOO sorrenc taa i MO Es dese Ko vla pele Baie ek an octo ALE nd eu cie ase ewe 24 Preparing the POR Master MIX 553 3 ia EE eda eE RE cea Phere NERONE Ro IO ds 24 Relative Quantification Getting Started Guide for the 7300 7500 System V Preparing the Reaction Plate 0 cc ee eee 25 Creating a Relative Quantification RQ Plate Document LLL 26 Specifying Thermal Cycling Conditions and Starting the Run 30 Analyzing and Viewing RQ Plate Data 0 cece 32 Exporting RO Plate D abd uu sate disi Duc a cent iac Eon es de RU ut a nh CER Beth doe ee hein Bee 34 Chapter5 Analyzing Data in an RQ Study 35 INK ON ass dap pat Barus tic eade ee dedos 4 osea dude ots eu es md eve ene eee 35 Creating an RQ Study Document 0 00 eens 36 Configuring Analysis Settings
33. e Threshold column displays AR IUE AS n Liver Aug 05 2003 02 the automatically calculated threshold values EE EE M FLT4 0 451420 Auto Auto The Auto Ct and Baseline columns are set to GAPSH lg 4i885 tka s A t 99 GTF2B 0 251278 Auto Auto uto GTF 2l 0 280024 Auto Auto HTR4 0 082686 Auto Auto i 1 1 KAI 0 323054 Auto Auto For more information about the settings in the Mr or Aado ae Analysis Settings dialog box refer to the Online LEER ae fo s 3 Help Se aaa s us Find gt a 1 A Sample Summary Well Information Add Plate Remove Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 39 Chapter 5 Analyzing 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 Adjusting 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 Threshold A typical amplification curve has a Plateau phase a Linear phase b Geometric phase c Background d Baseline e Experimental error such as contamination pipetting errors and so on can produce data that deviate significantly from data for typical amplification curves Such atyp
34. e 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 ROMin Max Confidence Level in the Analysis Settings dialog box see page 38 Notes 48 Absolute Quantification Getting Started Guide for the 7300 7500 System 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 Next 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 Jn Ki similarly if you want to change the baseline or ne threshold values the endogenous control the control type or the RQ Min Max parameters you need to reanalyze your data Bladder D S E S d ao Not
35. e specific nucleic acid probe reagent used for analyses performed on instruments To create a detector Detector Manager 1 Select Tools gt Detector Manager Note A plate document any type must be open 2003 0945 06 before you can access the Tools menu 2003 08 35 05 2003 09 30 13 2005095350 13 200509550 13 2003 08 50 13 nane RARA FAM none RMaseP3 FAM none RMaseP2 FAM none RNase P1 FAM none RNase F FAM none KAI FAM none 2003 0915 na IP Meterctor Internal Positive tli rmnnael 700a mA 18 ii gt 2 Select File gt New bul File i Add Ta Plate Document Mew Duplicate Add to Plate Document Import Export Clear Clear All Properties 3 Inthe New Detector dialog box enter a name for the detector Hew Detector Name e Description 4 n t a Quencher Dye nnd o Color Hi m Mates Create Another Cancel IMPORTANT The name of the detector must be unique and should reflect the target locus of the assay such as GAPDH or RNase P Do not use the same name for multiple detectors 4 Optionally click the Description field then enter a brief description of the detector Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 53 Appendix A 5 In the Reporter Dye and Quencher Dye drop down lists select the appropriate dyes f
36. ed 2 Verify that For two step RT PCR the default PCR thermal cycling conditions are set Forone step RT PCR you set the thermal cycling parameters as shown in Thermal Cycling Conditions for One Step RT PCR on page 30 e Sample volume is 50 uL e 9600 Emulation is selected Note If you are using SYBR Green I 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 The Al position is in the top left side of Well A1 the instrument tray 5 Click Start As the instrument performs the PCR run it displays real time status information in the Instrument tab and records the fluorescence 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 Absolute Quantification Getting Started Guide for the 7300 7500 System 31 Chapter 4 Generating Data from RQ Plates Analyzing and Viewing RQ Plate Data Analyzing and Viewing RQ Plate Data Starting the Analysis To analyze RQ
37. er Lee Uer Lee Liver Ler The figure on the right shows a completed plate a E z E E T set up H Lher Lee Uer Lee Uer Lee Uver Lee Uver Lee Uver Lie E E a i E i B H B H Notes 8 Relative Quantification Getting Started Guide for the 7300 7500 System 8 Start the RQ run a Select the Instrument tab By default the b standard PCR conditions for the PCR step of the two step RT PCR method are displayed Select File gt Save As enter a name for the RQ Plate document then click Save c Load the plate into the instrument d Click Start After the run a message indicates if the run is successful or if errors were encountered 9 Create an RQ Study document as described in Creating an RQ Study Document on page 36 Briefly a b Notes Select File gt New Select Relative Quantification ddCt Study in the Assay drop down list then click Next gt IMPORTANT RQ Studies are an optional add on for the 7300 instrument they are built in for the 7500 instrument Click Add to add plates to the study then click Open Note You can add up to 10 RQ plates to an RQ study Click Finish Example RQ Experiment Example RQ Experiment Procedure Hew Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Fielative Quantification ddCt Study C
38. eriment workflow is shown on page iii Notes 2 Relative Quantification Getting Started Guide for the 7300 7500 System RQ Studies with the 7300 7500 System Terms Used in Quantification Analysis Notes About RQ Experiments RQ Studies with the 7300 7500 System 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 The following figure illustrates the RQ Study process LULRL BUB I LE a d psp sd bladderplate sds Plated Reactions 7300 7500 System SDS Software RQ Plate Documents SDS Software RQ Study Document Note The 7300 7500 system software uses only the comparative method AAC to calculate relative quantities of a nucleic acid sequence 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
39. es Absolute Quantification Getting Started Guide for the 7300 7500 System 49 Chapter 5 Analyzing Data in an RQ Study 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 ug EB Bla ES fel mi Bins Ctvs Well Position Find al Plate Y mplification Ra 1 __ Detector Threshold Auto ct Baseline Auto 2 Inthe Data drop down list select Ct vs Well Position ACVRT 0 535276 Auto ACY RE COR 0 056001 3 In the RQ Detector grid select a detector to CD3D 0287214 Auto Auto E l FGF21 0 200000 examine All samples that use this detector are Eu TETTE GTFZB 0 251278 Auto Auto 35 GTFzl n 280024 Auto Auto HTR4 O 082666 Auta Auto a 4 Inthe RQ Samples grid click to select the displayed in the RQ Samples grid GAPDH 0 418936 Auto Auta samples to display in the Amplification Plot
40. ference dyes for all the wells on the plate ell Inspector Wiell s 41 44 Sample Mame Liver Detector Reporter Quencher Task Color EE m m RARA FAM none Target IMPORTANT If your experiment does not RR TaM f menes Taras use all the wells on a plate do not omit the M TACR2 FAM none Target Sr wells from use at this point You can omit L gom pe qe on o unused wells after the run For information N ee 000 0 about omitting unused wells refer to the Remove Close ROX Online Help Note You can change the sample setup information sample name detector task after a run is complete e Close the Well Inspector Notes 28 Absolute Quantification Getting Started Guide for the 7300 7500 System Creating a Relative Quantification RQ Plate Document Creating an 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 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 targe
41. g parameters the same number of steps cycles sample volume emulation mode The SDS software will reject a plate if it detects any differences The first plate added to the study serves as the reference plate against which other plates are compared 6 Click Finish If desired save the RQ Study document when prompted The SDS software opens a new RQ Study document and displays the RQ Study main view with its three panes a RQ Detector grid Allows you to select detectors to associate with the loaded study For each detector Color Detector name Threshold value Auto Ct and Baseline are displayed Note At this point all the values in the Threshold Auto Ct and Baseline columns are set to the default values 0 200000 Manual and 6 15 respectively b RQ Sample grid Displays the samples associated with the selected detector s The sample Grid displays numerical results of RQ calculations and has two subtabs sample Summary and Well Information Notes Creating an RQ Study Document Hew Dus unel Wicca h dd Flateg zu lates 1u Ih FQ Euy Select RO Plite 81 Dera imal libn d bly Compute he PMelisok 1e nen 1 Add Plates Hew Document Wizard Add Plates Add plates to the RQ Study Last Modified Bladder Kidney Liver d Remove Back Finish Cancel E Detector Threshold Auto CT 4 ACVRT D 200000 Ma
42. gnals check for nonspecific product formation using dissociation curve or gel analysis O e O O FORWARD PRIMER E NNNM O Q O o ST O REVERSE PRIMER Step 1 Reaction setup The SYBR Green dye fluoresces when bound to double stranded DNA Step 2 Denaturation When the DNA is denatured the SYBR Green dye is released and the fluorescence is drastically reduced Step 3 Polymerization During extension primers anneal and PCR product is generated Step 4 Polymerization completed SYBR Green I dye binds to the double stranded product resulting in a net increase in fluorescence detected by the instrument Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 15 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 Two step RT PCR is performed in two separate reactions first total RNA is reverse transcribed Two Step RT PCR 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
43. he 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 e Fold expression changes are calculated using the equation 27 CT Detector m Target amp E ae 8 Z E lt gt Q Q o Oo ue i A Sample Summary Well Information Detectors Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 45 Chapter 5 Analyzing Data in an RQ Study Analyzing and Viewing the Results of the RQ Study 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 clicking the y or x axes of a plot to display the Graph Settings dialog as shown on page 32 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
44. ic 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 Real Time PCR System Absolute Quantification Getting Started Guide PN 4347825 e Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Getting Started Guide PN 4347828 Applied Biosystems 7300 7500 Real Time PCR System Site Preparation Guide PN 4347823 e Sequence Detection 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 e Search through frequently asked questions FAQs e 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 Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mai
45. ical 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 43 Manual Baseline and Threshold Determination If you set the baseline and threshold values manually for any detector in the study you must perform the procedure on page 43 for each of the detectors The following amplification plots show the effects of baseline and threshold settings Notes 40 Absolute Quantification Getting Started Guide for the 7300 7500 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 E C im e a Baseline 1357 811131517 18 21 23 25 27 28 31 33 35 37 38 Cycle Number Baseline Set Too Low The amplification curve begins too far to the right of the maximum baseline Increase the End Cycle value c C m Z di e Baseline 357 8111315 17 19 21 23 25 27 29 31 33 35 37 38 1 Cycle Number Baseline Set Too High The amplification curve begins before the maximum baseline Decrease the End Cycle value Delta Rn Baseline s mg qo ee te Fal Ee be SED IgE X228 24 ate Be in es He E Cycle Number 1 Notes Absolu
46. idth lines 32 46 47 workflow RQ experiment overview 2 X x axis 32 46 47 Y y axis 32 46 47 60 Absolute Quantification Getting Started Guide for the 7300 7500 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 Bibbvstems Applera is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 01 2004 Part Number 4347824 Rev A an Applera business
47. ion Polymerase Activation HOLD HOLD CYCLE 2 min 50 C 10 min 95 C 15 sec 95 C 1 min 60 C Thermal Cycling Conditions for One Step Thermal Cycler Protacol Thermal Profile Auto Increment Hamp Rate RT PCR Stage 1 Stage 2 Stage 3 Reps Reps RepsEo If you select the one step RT PCR method cDNA Bs generation and amplification take place simultaneously at this point in the workflow The following table shows the thermal cycling conditions for one step RT PCR experiments Add Cycle Add Hold Add Step Add Dissociation Stage Note Refer to the Online Help for instructions on E i Sample Volume pL E i 9600 Emulation modifying thermal cycling parameters Amplification Plot Data Stage 3 Step 2 60 0 ce 1 00 m 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 Notes 30 Absolute Quantification Getting Started Guide for the 7300 7500 System Specifying Thermal Cycling Conditions and Starting the Run Thermal Cycling Conditions for One Step RT PCR To specify thermal cycling conditions and start the run 1 Select the Instrument tab By default the standard PCR conditions for the PCR step of the two step RT PCR method are display
48. ith a probe Specific hybridization between probe and target generates fluorescence signal e Provides multiplex capability e Optimized assays available e Allows 5 nuclease assay to be carried out during PCR Polymerization FORWARD R REPORTER PRIMER R PROBE Q E 3 Q QUENCHER 7 Qa REVERSE PRIMER Step 1 A reporter R and a quencher Q are attached to the 5 and 3 ends of a TaqMan probe Cleavage z S 9 3 Step 2 During each extension cycle the AmpliTaq Gold DNA polymerase cleaves the reporter dye from the probe 5 o wy 5 Strand Displacement 9 a Qa AR q Step 1 continued when both dyes are attached to the probe reporter dye emission is quenched Polymerization Completed Q zs 9 2 a Qa 5 3 5 Step 3 After being separated from the quencher the reporter dye emits its characteristic fluorescence SYBR Green I reagents Description Uses SYBR Green dye a double stranded DNA binding dye to detect PCR products as they accumulate during PCR cycles Advantages e Reduces cost no probe needed e Amplifies all double stranded DNA e Yields a melting profile of distinct PCR runs e Increases sensitivity for detecting amplification products relative to product length Limitations Binds nonspecifically to all double stranded DNA sequences To avoid false positive si
49. ix 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 chemical N eue 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 N M blhiel Indicates a potentially hazardous situation that if not avoided could result in death or serious injury Relative Quantification Getting Started Guide for the 7300 7500 System vii Preface How to Obtain More Information Safety Refer to the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Getting Started Guide and the Applied Biosystems 7300 7500 Real Time PCR System Site Preparation Guide for important safety information How to Obtain More Information For more information about using the 7300 7500 system refer to e Applied Biosystems 7300 7500 Real Time PCR System Online Help e Applied Biosystems 7300 7500 Real Time PCR System Allel
50. justing 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 Analyzing and Viewing the Results of the RQ Study fe Omit Data Delta Rn vs Cycle Calibrator iver Orientation Gene Expression Plot Graph Settings Bar width Hi z 0 100 Group Spacing 70 0 100 Group Spacing only applicable if B ar Width is 10605 3D Bars Sample Detector Mame In Bar W Auto Scale Minimum Masimum wa C emn Y Ass Data Logg AO Raw AG Defaults Detector 7 Plate Y Amplification Plot Y Gene Expression Log 10 Relative Quantification 0 20 0 Imp E g e E g CCR2 CD3D FLT4 GAPDH Detectors Absolute Quantification Getting Started Guide for the 7300 7500 System 47 Chapter 5 Analyzing Data in an RQ Study Analyzing and Viewing the Results of the RQ Study Gene Expression Plot Orientation Sample E omit Deta Deta Rnvs Cycle Calibretor Liver Orientation Plate Y Amplification Plot Y Gene Expression samples are plotted on the x axis and each bar shows the set of sample values of a single detector emma c o u m o c Iud pu E Im a a e q D saj Kidney Liver Error Bars for Gene Expression Plots The SDS software displays error bars for each column in th
51. kground Below the plateaued and linear regions of the amplification curve Within the geometric phase of the amplification curve The SDS software adjusts the theshold value and displays it in the Threshold field after reanalyzing 5 Repeat steps 2 through 3 to set the baseline and Delta Rn s Cycle 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 i a a my 2 11 13 18 Jr 13 21 23 285 arp ai al 33 29 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 Notes 44 Absolute Quantification Getting Started Guide for the 7300 7500 System Analyzing and Viewing the Results of the RQ Study Selecting Detectors to Include in Results Graphs Analyzing and Viewing the Results of the RQ Study Selecting Detectors to Include in Results Graphs In the RQ Detector Grid select detectors to include in the result graphs by clicking a detector Ctrl click to include multiple detectors Click drag to include 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 Plot or Gene Expression analysis results are displayed To see information about a specific well select the Well Information tab
52. l 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 SDS v1 2 Software 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 Chi ck OK in place sample name editing Feature select well s and type text directly into the sample name After editing sample name s you can then save the RQ Plate document The SDS software displays the Well Inspector f Enter the sample names in the Well Inspector ye a f De View gt Well Inspector oaa ERE Tn ERE se EE ES ERE RN ERE RE REL En X Liver Lher Uvar Lher Uvar Lher Uvar Lher Uvar Lher Uvar Lher a H E El El z IMPORTANT If your experiment does not use an H uH H T B W HB mW all the wells on a plate do not omit the wells Cum uber uw Uver Lwe Uber LNe Uber Ne Uer Lee 5 his point Y TRAMA 8 H E B H B 8 m 5 m rom use att i point ou can omi UH Ne S B Over Leer Uver Liver ger LNer Liver Lee Uver Lwe Liver Liver after the run is completed For more information m liver E about omitting wells refer to the Online Help an Su ia M ne e ue Mr Dor erus H H E m E Bi a Bi Uvar Lher Uvar Lher Uvar Lher Uvar Lher Uvar Lher Uvar Lher 1 G O Lee Liver Lee Uver Lee Liv
53. l your comments to techpubs appliedbiosystems com viii Relative Quantification Getting Started Guide for the 7300 7500 System Chapter 1 Overview Introduction and Example RQ Experiment Notes Introduction and Example RQ Experiment About the 7300 7500 system See page 2 About relative quantification See page 2 About RQ experiments See page 2 About the example RQ experiment dio Relative Quantification Getting Started Guide for the 7300 7500 System Chapter 1 Introduction and Example RQ Experiment About the 7300 7500 System About the 7300 7500 System Description The Applied Biosystems 7300 7500 Real Time PCR System 7300 7500 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 Relative The 7300 7500 system allows you to perform several assay types using plates or tubes in Quantification the 96 well format This guide describes the relative quantification RQ assay type Assay For information about the other assay types refer to the Sequence Detection Systems Chemistry Guide SDS Chemistry Guide and the Online Help for the 7300 7500 system Online Help About Relative Quantification Definition Relative quantifica
54. mand products 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 1f 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 54 Absolute Quantification Getting Started Guide for the 7300 7500 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 Absolute Quantification Getting Started Guide for the 7300 7500 System 55 References 56 Absolute Quantification Getting Started Guide for the 7300 7500 System Numerics
55. n Each primer pair amplifies either a target sequence or an endogenous control L m I3 Target Primer Set L OO C Endogenous Control wi Wi 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 Type of chemistry you use to detect PCR products Singleplex PCR can use either SYBR Green or TagMan reagent based chemistry Multiplex PCR can use only TaqMan chemistry 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 multiplex PCR increases throughput and reduces the effects of pipetting errors For more information about multiplex and singleplex PCR refer to the SDS Chemistry Guide PN 4348358 Example Experiment The si
56. ng 53 definition 53 selecting for RQ studies 38 deviation standard 42 display options 32 46 47 documentation feedback vili documents exporting 34 52 RQ Plate 26 RQ Study 36 templates 27 dyes FAM 17 54 reporter 3 ROX 28 33 Absolute Quantification Getting Started Guide for the 7300 7500 System 5 Index dyes continued SYBR Green 15 17 TAMRA 33 54 E emulation mode 9600 31 endogenous controls associating with detectors 26 definition 13 for RQ plates 26 selecting for RQ studies 39 endpoint PCR 2 equipment 4 example RQ experiment components 14 creating detectors 54 description 5 PCR master mix 25 PCR method 12 reverse transcription 22 RQ Plate document example 29 RQ Study document example 45 exporting data RQ plates 34 RQ studies 52 F FAM dye 17 54 G Gene Expression plots 47 geometric phase of amplification curve 40 graph settings 32 46 47 guidelines preparing RNA 20 H High Capacity cDNA Archive kit 21 importing plate setup information 27 Instrument tab 31 L line width 32 46 47 linear phase of amplification curve 40 M master mix PCR 24 08 materials 4 mode emulation 31 MSDSs obtaining viii multiplex PCR 12 N New Detector dialog box 53 normalized reporter 3 O options display 32 46 47 options graph 32 46 47 outliers 50 P passive reference 3 28 PCR end point 2 master mix preparing 24 multiplex 12 real time 2 selecting a method
57. ngleplex 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 12 Absolute Quantification Getting Started Guide for the 7300 7500 System Specifying the Components of an RQ Experiment SS Specifying the Components of an RQ Experiment After you decide to use the singleplex or multiplex method you need to specify the required components of the RQ experiment for every sample A target The nucleic acid sequence that you are studying A calibrator The sample used as the basis for comparative results 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
58. ngs Post Run Settings Y xis Auto Scale Minimums Y xis v Auto Scale Linear Minimum 0 0001 Linear Minimum fe Log ie Log Maximum 10 M asinum Axis is autoscaled in RealTime Axis v Auto Scale t Minimum Ke BE ee Maximum Display Options Line Width 2 1 10 Defaults Cancel Apply 32 Absolute Quantification Getting Started Guide for the 7300 7500 System Plate Tab Displays the results data of each well including the e Sample name and detector task and color for each well Calculated R value Spectra Tab Displays the fluorescence spectra of selected wells The Cycles slider allows you to see the spectra for each cycle by dragging it with the pointer 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 Pp ore d EAN Y Spectra Y f Component Plot 6 42 B Liver 587 C Liver 6 51 D Liver 5 94 E Liver B 75 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 H 7 0 Setup Y Instrument Y Results Plate Y Spectra Y Component Y Amplification Plot 9220 8000 6000 4000
59. nt m t uL 5 Reactions Final Concentration TagMan Universal PCR Master Mix 2X 25 0 125 0 1X 20X Assays on Demand Gene 2 5 12 5 1X Expression Assay Mix cDNA sample 5 0 25 0 50 ng for the 50 uL reaction Nuclease free water 17 5 87 5 Total 50 0 250 to account for pipetting losses CDNA are added to each well eoooooooooo200 eoeooooooooooe0200 000000000000 0 0 0 0 0 0 0 0 0 90 09 eoooeoooooooo0000 eoocco0o000000 0000000000097 Endogenous 0 0 0 0 0 0 9 0 O O O O controls GAPDH eooooooeooooo200 eoeoooooooooo000 eeoeoooooooooooo0200 Kidney ececc000000090 samples eooooeooooooooo00200 eooooooooooooo00 0 006000000000909 Endogenous 9 9 0 0 0 0 9 0 O O OF 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 000800000008 0000080080080 TALLILLE 000000800008 0000080800008 0000080080000 000008080800080 eeeeeee e0ood The reactions are kept on ice until the plates are loaded on the 7300 7500 system Samples and endogenous controls are arranged on three plates as shown below 50 uL of PCR master mix containing Bladder samples Endogenous controls GAPDH Notes Absolute Quantification Getting Started Guide for the 7300 7500 Sy
60. nual ACVR2 D 200000 Manual COR2Z unn Manual CD3D DoD Manual FGF21 D 200000 Manual FLT4 0 200000 Manual GAPDH 0 200000 Manual GTF2B D 200000 Manual GTF2l D 200000 Manual HTR4 D 200000 Manual KA D 200000 Manual MCAMT D 200000 Manual MFA 0 200000 Manual 3 mee lll B o gt Eind B Sample Detector 4 lii gt A Sample Summary A Well Information j TM a7dur sda doep sda T1 er i72 m E iez fce Rn Fhris Add Plate Look n o 33zete e of Ea z i E Mu Fel E il eee Open Open EGE E3 Aug 05 2003 02 04 PM Aug 05 2003 02 05 PM Aug 05 2003 02 05 PM E Omit Data Delta Rin vs Cycle Calik Find P V Amplification Plot Gene Expression Plate Hame Last Modified 1 Bladder Aug 05 2003 02 04 PM 2 Kidney Aug 05 2003 02 05 PM 3 Liver Aug 05 2003 02 05 PM Remove Absolute Quantification Getting Started Guide for the 7300 7500 System 37 Chapter 5 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
61. o tasks to each detector in each well of a plate document Task Symbol Apply to detectors of Target Wells that contain PCR reagents for the amplification of target sequences Endogenous B Wells that contain reagents for the amplification of the Control endogenous control sequence 26 Absolute Quantification Getting Started Guide for the 7300 7500 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 7300 7500 gt Applied Biosystems 7300 7500 SDS Software Fey to start the SDS software 2 Select File gt New 3 Inthe 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 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
62. ocess Further 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
63. om 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 different 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 Experiments can also be designed 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 eooooooooooooe02000 i H i N ecc000000000 0090000000000 0 0 0 1008 06 090 O eeee0606000000 eooooeooooooo00e0200 samples eeeoecc000000 eoeoooo00000
64. ontainer 96 Wwell Clear Template Blank Document Browse Operator Administrator Comments Z Default Flate Name Plate1 5 amp Back Finish Cancel Relative Quantification Getting Started Guide for the 7300 7500 System 9 Example RQ Experiment 10 Analyze the RQ data as explained in Chapter 5 a Configure analysis settings 1 using the Auto Ct option and analyze the data Note See Configuring Analysis Settings on page 38 for details If you know the optimal baseline and threshold settings for your experiment you can use the Manual Ct and Manual Baseline options b If necessary manually adjust the baseline and threshold Note See Adjusting the Baseline and Threshold on page 40 c Click amp or select Analysis gt Analyze to reanalyze the data d View analysis results by clicking a tab in the 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 Notes Chapter 1 Introduction and Example RQ Experiment Analysis Settings Relative Quantification Ct Analysis Detector All Auto Ct Manual Ct Threshold 200000 z d Start cycle Auto End cycle Auto Calbrator Sampe Endogenous Control Detector GAPDH Control Type Gene Expre
65. or 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 6 Click the Color box select a color to represent the detector using the Color dialog box then click OK f Optionally click the Notes field then enter any additional comments for the detector 8 Click OK to save the detector and return to the Detector Manager 9 Repeat steps 2 through 8 for the remaining detectors 10 In the Detector Manager click Done when you finish adding detectors B Example Experiment In the example RQ experiment 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 ACVH1 and assigned a yellow color Because all Assays on Demand products have probes that are labeled with FAM dye FAM was selected for the reporter dye Additionally Assays on Demand products use TaqMan MGB probes which use nonfluorescent quenchers No quencher dye is selected for the detector Note Assays on De
66. ported 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 34 Absolute Quantification Getting Started Guide for the 7300 7500 System Chapter 5 Analyzing Data in an RQ Study Workflow Create a new See page 36 RQ Study document RS Configure analysis settings See page 38 Adjust the See page 40 baseline and threshold Analyze and view results See page 45 If necessary S 50 omit samples pps Export the RQ Study document if desired See page 52 Performing an RQ Study Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 35 Chapter 5 Analyzing Data in an RQ Study Creating an RQ Study Document Creating an RQ Study Document To conduct a comparative analysis of RQ plates in a study 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 instrument The SDS software uses the comparative method 2 AC 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 control type
67. r 5 0 50 to 900 nM Reverse primer 5 0 50 to 900 nM TaqMan probe 5 0 50 to 250 nM cDNA sample 5 0 10 to 100 ng Nuclease free water 5 0 Total 50 0 If you design probes and primers using Primer Express software they must be optimized to work with the universal assay conditions using the volumes listed in the table above All Assays by Design and Assays on Demand products are formulated so that the final concentration of the primers and probes are within the recommended values Notes 24 Absolute Quantification Getting Started Guide for the 7300 7500 System Preparing the Reaction Plate Preparing 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 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 Example Experiment Primers and probes for the example RQ experiment are obtained from the Assays on Demand product line and are provided as a 20X Gene Expression Assay Mix The PCR master mix is prepared as follows Reaction Compone
68. ree RQ plates one for each tissue All three plates are analyzed in an RQ study with the liver samples serving as the calibrator Relative Quantification Getting Started Guide for the 7300 7500 System Example RQ Experiment Example RQ Experiment Procedure 1 Design the experiment as explained in Chapter 2 a Designate the targets calibrator endogenous control and replicates b Order the reagents for TaqMan probe based chemistry C Order the appropriate Assays on Demand 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 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 NE 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 Chapter 1 Introduction and Example RQ Experiment
69. scribed in Chapter 4 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 5 Prepare the reaction plates a Label the reaction plates ensuring that you include an endogenous control on each plate b Pipette 50 uL of the appropriate PCR master mix containing cDNA into each well of the plate c Keep the reaction plates on ice until you are ready to load them into the 7300 7500 system Notes Example RQ Experiment Example RQ Experiment Procedure Step Type Time Temperature HOLD 10 min 25 C HOLD 120 min 37 C PCR Master Mix Final Reaction uL uL 5 C b oncen Component Sample Reactions i tration TaqMan Universal 25 0 125 0 1X PCR Master Mix 2X 20X Assays on 25 12 5 1X Demand Gene Expression Assay Mix CDNA sample 5 0 25 0 10 to 100 ng Nuclease free water 175 87 5 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 ececc00000000 ececcc060060000 000000000000
70. ssays by Design products probes are already labeled with a reporter dye If you design your own assays you need to specify a reporter dye for your custom probe s For singleplex experiments you can use the same dye for targets and endogenous control s For multiplex experiments the probe for the target 1s typically labeled with FAM dye and that for the endogenous control with VIC dye Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 17 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 Assays on Demand products 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 ACVH1 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
71. ssion Mon multiplexed RU Min Max Confidence 35 00 He Replicate and Outlier Removal Iv Remove Outlier OK amp Reanalyze Cancel Apply Threshold falls within geometric phase of curve Baseline is set before amplification begins Jc E 44 84 E Omit Data Delta Rn vs Cycle v Calibrator Liver a Plate Y Amplification Plot Y Gene Expression 0 20 Orientation Detector Log 10 Relative Quantification lt gt N Sample Summary Well Information 10 Relative Quantification Getting Started Guide for the 7300 7500 System Chapter 2 Designing an RQ Experiment Workflow Notes Designing an RQ Experiment Select the See page 12 PCR method Specify the components of an RQ experiment oes es Select the chemistry See page 15 Select one step or See page 16 two step RI PCR Choose probes See page 17 and primers Absolute Quantification Getting Started Guide for the 7300 7500 System 11 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 Asingleplex reaction where a single primer pair 1s 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 reactio
72. stem 25 Chapter 4 Generating Data from RQ Plates Creating a Relative Quantification RQ Plate Document 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 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 as defined in Specifying the Components of an RQ Experiment on page 13 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 tw
73. t 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 Detector task and color Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 29 Chapter 4 Generating Data from RQ Plates Specifying Thermal Cycling Conditions and Starting the Run Specifying Thermal Cycling Conditions and Starting the Run Default Thermal Cycling Conditions for hemal Lucler Protocol Thermal Profile Auto Increment Hamp Rate PC R Stage 1 Stage 2 Stage 3 Reps Reps RepsEo If you selected the two step RT PCR method for your BS RQ experiment recommended you have already completed the RT step and are ready to PCR amplify cDNA The default thermal cycling conditions for the PCR mr i cle o e Issociation Stage step of the procedure shown in the following table eT ES i ee gt Settings should appear in the Instrument tab Sample Volume ul 90 IV 9600 Emulation Amplification Plot Data Stage 3 Step 2 60 0 ce 1 00 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 Activat
74. te Quantification Getting Started Guide for the 7300 7500 System 41 Chapter 5 Analyzing Data in an RQ Study Adjusting the Baseline and Threshold Threshold Set Correctly The threshold is set in the geometric phase of the amplification curve Threshold settings above or below the optimum increase the standard error of the replicate groups 0 000 1 000 1 000 0 828 1 208 2 008 0 249 0 002 0175 0 353 n 3 047 0 070 0 002 0 050 0 096 O 0 111 0 207 0 193 CD3D CD3D Liver Target Target Target 31 585 10 216 Kidney Bladder CD3D Threshold Set Too Low The threshold is set below the geometric 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 geometric phase of the curve 24 1417 0 006 21 5141 658 24 043 2 673 0 246 0 264 0 281 CD3D CD3D Liver Target Target Target 0 000 1 000 1 000 0 659 1 517 1 694 0 309 0 002 i n 2 679 0 156 0 002 0 097 Kidney Bladder CD30 Threshold Set Too High The threshold is set above the geometric 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 geometric phase of the curve Sample Detector Task 0 000 2 340 4617 D D41 0 002 D D24 1 000 1 000 0 824 1 2144 0 0 197 0 002 0 092 0 423 0 n
75. terest are available from the Assays on Demand product line which uses TaqMan chemistry Two step RI PCR is performed using the reagents recommended for TaqMan reagent or kit based chemistry in the table above Choosing the Probes and Primers Choose probe and primer sets for both your target and endogenous control sequences Applied Biosystems provides three options for choosing primers and probes e Assays on Demand Gene Expression Products Provide you with optimized ready to use TaqMan 5 nuclease assays for human mouse or rat transcripts For information on available primer probe sets go to http www allgenes com e Assays by Design Service 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 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 v2 0 User s Manual PN 4329500 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 Assays on Demand or A
76. tion 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 Real time PCR RQ is performed using real time PCR In real time PCR assays you monitor the Assays 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 1s 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 About RQ Experiments RQ Experiment In this document the term RQ experiment refers to the entire process of relative Workflow quantification beginning with generating cDNA from RNA reverse transcription and ending with analyzing an RQ study The RQ exp
77. uto Ct f Manual Ct Threshald D 267214 5 Start cycle Auta End cycle Auta Sample summary Well Information F 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 displays the Associated samples from all plates included in the study in the RQ sample grid 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 3 Set the baseline for the detector a Under Analysis Settings select Manual Baseline Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 43 Chapter 5 Analyzing Data in an RQ Study Adjusting the Baseline and Threshold 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 gt 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 Above the bac
78. when applicable e Set baseline and threshold values and RQ Min Max Confidence Levels e Omit individual wells or sample replicates e Select the endogenous control and the calibrator sample e Create add or modify samples e Create add or modify detectors e Change detector tasks You can perform these operations in RQ Plate documents To create a new RQ Study document 1 Select File New 2 In the Assay drop down list of the New Document Wizard select Relative Quantification dd Ct Study Accept the default settings for Container and Template 96 Well Clear and Blank Document 3 Enter a name in the Default Plate Name field or accept the default 4 Click Next gt Notes 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 Container 36 w ell Clear m Template Operator Administrator Comments Default Plate Mame PPlate15 Finish Cancel 36 Absolute Quantification Getting Started Guide for the 7300 7500 System 5 Add RQ plates to the study a Click Add Plates Note You can add up to 10 RQ plates to an RQ study b Select the plate s that you want to add to the study then click Open The selected plates are displayed IMPORTANT All plates added to a study must have identical thermal cyclin
79. ystems 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 30 Notes Absolute Quantification Getting Started Guide for the 7300 7500 System 21 lt 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 Nara 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 For the example experiment RNA is extracted from the liver bladder and kidney tissues of an individual RNA concentration is determined spectrophotometrically using Asso and the 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

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