Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

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Chapter 6Analyzing Real-Time DataEssential Experimental ComponentsEndogenousControlCalibratorSampleAll relative quantification experiments require data from a second probe and primerset that amplifies an endogenous control sequence. Endogenous control targets aretypically constitutive RNA or DNA sequences that are present at a statisticallyconsistent level in all experimental samples. By using the endogenous control as anactive reference, the data from the amplification of a messenger RNA (mRNA) targetcan be normalized for differences in the amount of total RNA added to each reaction.Examples of common endogenous controls are: GAPDH, 18S rRNA, and β-Actin.The endogenous control can be amplified independently of the target sequences inseparate wells on the reaction plate (as singleplex or non-multiplex reactions), or inthe same well (as multiplex reactions).IMPORTANT! For non-multiplex experiments, the reactions amplifying theendogenous control must be located on the same plate as the target assays.IMPORTANT! To generate a standard deviation for the relative quantity value of atarget, each plate must contain usable data from at least two replicates of the targetand endogenous control.All relative quantification experiments require data from a calibrator sample. Duringanalysis, the SDS software calculates gene expression levels in samples relative tothe level of expression in a calibrator sample. Thus, the calibrator plays an integralpart in the calculation because it is used as the basis for the comparative results.Examples of possible calibrator samples include:• A zero time-point sample in a time-course experiment• An untreated sample (versus treated samples)• A resting sample (versus activated samples)Note: The SDS software combines the data from replicate calibrator wells at the ∆C Tlevel of the relative quantification calculation (whether the replicates are present on asingle or multiple plates).Replicate WellsFor relative quantification studies, Applied Biosystems recommends the use of threeor more replicate reactions per sample to ensure statistical confidence.Replicates allow you to:• Preserve Data – If an amplification fails in one well, replicate wells canpotentially provide data. This point is especially true in the case of endogenouscontrols which, upon failure, may invalidate the results for the entire plate.• Remove Outliers – The use of replicate populations provide the opportunity forthe visualization and removal of outliers.• Ensure Statistical Reproducibility – In general, the use of replicates ensures agreater degree of experimental reproducibility by providing a means to identifyand refute anomalous data caused by experimental error (such as contamination,pipetting errors, and so on).6-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm
Getting StartedGetting StartedUsing the SDSSoftwareOnline HelpExamples in ThisChapterFor specific instructions on any procedure described within this section, refer to theSequence Detection Systems Software Online Help. To get help at any time during theprocedure, click located within the dialog box or window in which you areworking.The screenshots that appear within this chapter were created using a series ofTaqMan ® Cytokine Gene Expression Plates (PN 4304671), a research tool forreal-time, in vitro quantitative evaluation of human cytokine gene expression. Theplate detects the expression of 12 cytokine target sequences and an endogenouscontrol in complementary DNA (cDNA) samples. The TaqMan Cytokine GeneExpression Plate I consists of a MicroAmp ® Optical 96-Well Reaction Plate arrangedinto 24 replicate groups, each containing TaqMan probes and primers for the assay ofone human cytokine mRNA and an endogenous control. Figure 6-8 illustrates theassay configurations for the plate.The study used to produce the screenshots for this section consisted of four plates ofa time-course experiment run to evaluate the expression of 24 target cytokine mRNAover a 36-hour period.36 hrIL-2IL-1αIL-1βPlate 1IL-3 IL-4 IL-5IL-6 IL-7 IL-8Plate 2GR210824 hrIL-10 IL-12p35 IL-12p40GR2108IL-13IL-15IL-17IL-18 G-CSF GM-CSFPlate 312 hrM-CSF IFNγ LTβGR21080 hrTGFβ TNFα TNFΒGR2108Plate 4GR2108Figure 6-8Sample Configuration of the Example PlatesNote: For more information on the TaqMan Cytokine Gene Expression Plate I, seethe TaqMan Cytokine Gene Expression Plate I Protocol (PN 4306744).Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-21DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm
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Applied Biosystems7900HT Fast Real-
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ContentsPrefaceHow to Use This Guid
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Chapter 4Operating the InstrumentNo
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Chapter 7Maintaining the Instrument
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Appendix D Theory of OperationFluor
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PrefaceHow to Use This GuidePurpose
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Safety and EMC Compliance Informati
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Symbols on InstrumentsSymbols on In
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General Instrument SafetyGeneral In
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Chemical Waste SafetyChemical Safet
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Physical Hazard SafetyOvervoltageRa
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Workstation SafetyWorkstation Safet
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Product Overview 11In This Chapter
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7900HT FAST Real-Time PCR SystemSec
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7900HT InstrumentInterchangeableThe
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7900HT FAST Real-Time PCR SystemBar
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Compatible ConsumablesCompatible Co
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Instrument ConnectionsFixed-Positio
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Section 1.2 Getting to Know the Sof
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SDS Software Related Files and Form
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7900HT FAST Real-Time PCR SystemMan
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Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
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Section 1.3 SDS Enterprise Database
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About the SDS Enterprise Database S
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Getting Started 22In This Chapter G
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Getting StartedUsing the SDSSoftwar
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GR23967900HTPowering On the 7900HT
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Using the SDS SoftwareUsing the SDS
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Using the SDS SoftwareAbout theMess
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Basic Software Skills TutorialNotes
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Basic Software Skills TutorialExerc
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Basic Software Skills TutorialExpor
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Basic Software Skills TutorialLesso
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Basic Software Skills Tutorial3. Se
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Using SDS Plate DocumentsUsing SDS
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Using SDS Plate DocumentsWell Inspe
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Preparing a Run 33In This Chapter N
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Notes for Database UsersDescription
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Workflow OverviewWorkflow OverviewE
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Workflow OverviewAllelicDiscriminat
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Step 1 - Creating a Plate DocumentS
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Step 2 - Applying Detectors and Mar
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Step 3 - Configuring the Plate Docu
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Step 5 - Programming the Plate Docu
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Step 6 - Saving the Plate Document
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Step 7 - Creating a Plate Document
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Step 9 - Running the Plate on the 7
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Operating the Instrument 44In This
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Notes for Database UsersDescription
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Section 4.1 Consumable PreparationS
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Compatible ConsumablesTable 4-1Comp
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Preparing Optical Plates for Usea.
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Preparing TaqMan Low Density Arrays
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Section 4.2 Running an Individual P
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Running a Single Plate (Using the S
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Running a Single Plate (Using the S
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After the RunAfter the RunUser Acce
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Section 4.3 Automated OperationSect
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Page 165 and 166: OverviewTable 5-1Signal and Sequenc
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Page 169 and 170: Opening the Run DataOpening the Run
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Page 177 and 178: After the AnalysisSaving theResults
Page 179 and 180: Analyzing Real-Time Data 66In This
Page 181 and 182: Notes for Database UsersDescription
Page 183 and 184: Section 6.1 Absolute Quantification
Page 185 and 186: OverviewAlgorithmicManipulation ofR
Page 187 and 188: Analysis ChecklistAnalysis Checklis
Page 189 and 190: Analyzing the DataAnalyzing the Dat
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Page 193 and 194: Section 6.2 Relative Quantification
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Page 203 and 204: Creating the Studyd. In the Plate Q
Page 205 and 206: Analyzing the Study• Manual Ct -
Page 207 and 208: Analyzing the StudySpecifying the A
Page 209 and 210: Viewing ResultsViewing ResultsDispl
Page 211 and 212: Viewing ResultsAbout DownArrowsDown
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Page 217 and 218: Before You BeginExample ResultsFigu
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Page 225 and 226: Setting the Baseline and Threshold
Page 227 and 228: Eliminating Outlying AmplificationE
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Performing a Background RunPreparin
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Performing a Background RunAnalyzin
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Performing a Pure Dye RunAfter a ru
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Performing a Pure Dye Run• Fast 9
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Performing a Pure Dye RunAnalyzing
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Adding Custom Dyes to the Pure Dye
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Adding Custom Dyes to the Pure Dye
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Verifying Instrument PerformanceTab
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Verifying Instrument PerformancePre
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Section 7.2 Maintaining the Plate H
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Adjusting the Sensitivity of the Pl
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Adjusting the Sensitivity of the Pl
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7900HT FAST Real-Time PCR SystemAli
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Aligning the Plate Handler5. Using
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Aligning the Plate Handler7. Using
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Aligning the Plate Handler13. Click
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7900HT FAST Real-Time PCR SystemAli
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Aligning the Fixed-Position Bar Cod
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Section 7.3 Maintaining the Compute
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Maintaining the SDS softwareSeveral
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Troubleshooting 88In This Chapter T
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Troubleshooting TableTable 8-1Troub
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Low Precision or IrreproducibilityL
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Low Precision or IrreproducibilityD
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Background RunsBackground RunsBackg
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Pure Dye Runs6. Repeat step 4 until
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End-Point Runs (Allelic Discriminat
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Software and 7900HT InstrumentTable
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Zymark Twister Microplate Handler a
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TaqMan Low Density ArrayTable 8-6Tr
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Software ReferenceAAIn This Appendi
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Importing Plate Document Setup Tabl
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Setup Table Files1. File VersionDes
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Setup Table Files6. Detectors ListD
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Setup Table Files9. Markers List (A
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Setup Table FilesFormat(for a singl
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Setup Table FilesFormat(for a singl
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Setup Table FilesExample Code A-16A
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Exporting Plate Document Data4. In
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Operating the SDS Software from a C
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Using the Search ToolLogical Operat
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Connecting SDS Software to the Data
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Designing TaqMan Reagent-BasedAssay
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Assay Development Guidelines• For
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Design Tips for Allelic Discriminat
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Kits, Reagents, and ConsumablesCCIn
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Consumables and DisposablesConsumab
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Instrument Maintenance and Verifica
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Custom Oligonucleotide SynthesisTab
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Theory of OperationDDIn This Append
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Fluorescent-Based ChemistriesBasics
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Real-Time Data AnalysisPhase 2: Lin
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Real-Time Data AnalysisSignificance
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A similar equation for the endogeno
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Limited Warranty StatementEEWarrant
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BibliographyGeneral Paperson TaqMan
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Klein, D., Janda, P., Steinborn, R.
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Dolken, L., F. , Schuler, and G. ,
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AllelicDiscriminationVirtanen M., H
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Glossary.sdd.sdm.sds.sdt∆C T∆
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ackgroundbaselinecalibratorcDNA(com
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minor groovebinder (MGB)multicompon
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R nrunsessionsingle nucleotidepolym
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IndexSymbols- 6-31↑ 6-33Numerics7
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fluorescent, common sources 8-7isol
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adjusting step parameters 3-21confi
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sample block locking bar 7-8sample
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Worldwide Sales and SupportApplied
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