QPCRQPCR ApplicationsApplications usingusing StratageneStratagene’’ss MxMx RealReal--TimeTime PCRPCR PlatformPlatform

Dan Schoeffner, Ph.D Field Applications Scientist [email protected] Tech. Services 800-894-1304 PolymerasePolymerase DNA

ChainChain Melt Anneal primers Gene of ReactionReaction interest (Amplicon)

Extension/Measure Forward and Reverse Primers

Melt Anneal

+ QQPCRPCR MolecularMolecular MechanismMechanism

Exponential amplification of the original DNA sequence (template) to create copies of part of the sequence (amplicon)

n n Xn=X0 (1+E)2 X = DNA concentration X0= Starting DNA concentration Xn= DNA concentration at cycle n E = Efficiency of PCR reaction, 0-1

http://allserv.rug.ac.be/~avierstr/principles/pcr.html InfluenceInfluence ofof ReactionReaction EfficiencyEfficiency TypicalTypical PCRPCR AmplificationAmplification PlotPlot

n o ti a c fi li p Baseline m A Raw Signal (R) Fluorescence (R) Threshold

Ct Cycle # CCtt = Fractional PCR cycle number at which the fluorescence intensity crosses the established threshold line.

• A 1Ct difference between samples represents 2x more transcript WhyWhy isis QPCRQPCR superiorsuperior toto PCRPCR

96 technical replicates

Variability using Variability using QPCR endpoint PCR GelGel--basedbased quantificationquantification

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1 2 RealReal--timetime quantificationquantification

1 2 UnpredicableUnpredicable AmplificationAmplification PlotsPlots withwith EndpointEndpoint AnalysisAnalysis ChemistriesChemistries usedused inin QPCRQPCR

TETTET HEXHEX TAMTAM TxRdTxRd FAMFAM JOEJOECy3Cy3 ROXROX Cy5Cy5 Alx350Alx350

350nm 700nm λ

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s Ab λ Light Fluorescence Detection Fluorescence Detection QuantitativeQuantitative PCRPCR ChemistriesChemistries dsDNA Binding SYBR Green

Probe Based TaqMan® Detection Molecular Beacons Lux® primers Hybridization probes ScorpionsTM Amplifluor® probes FRET ChemistriesChemistries

SYBR green dsDNA binding dyes Pro: ■ Ease of use ■ Inexpensive ■ Good for high throughput screenings Æ lots of genes: this is your chemistry ■ Great for first screens and optimization ■ Can detect amplicon heterogenity

Con: ■ Sequence unspecific – detects any double strand in your reaction ■ Can not multiplex reactions 1000x increase in fluorescence PrimerPrimer SelectionSelection

• Try to achieve similar Tm for all primers: Ideal ~60°C. (Future multiplexing or use of Taqman™ assays in mind)

• Forward and reverse primer should have ∆Tm <2°C

• 40-60% GC content to prevent G/C region self-hybridization

• ∆G of primer dimer/cross primer dimer formation > -4 kcal/mol to avoid stable primer dimers

• Design via software (Always use the same one):

• Always perform a BLAST search with your amplicon and primers (Æ Specificity of the PCR) SYBRSYBR greengreen

Raw Fluorescence [R]

SYBR Green I™ Thermal Profile

Negative First Derivative [-R’(T)]

Activation Amplification Dissociation ChemistriesChemistries

Taqman probes Pro: R ■ Sequence specific Q ■ Possibility to do multiplex - have GOI and normalizer in the same well, doing comparative quantification Taq R Q

Con: R ■ More difficult to design ■ Expensive Q Taq LinearLinear TaqmanTaqman ProbeProbe DesignDesign

•Probe Tm 5-10°C higher than primers • ≤ 30 bp in length • No G next to reporter fluorophore • < 4 contiguous Gs • PCR blocker at 3’ end • Compatible reporters and quenchers LinearLinear TaqmanTaqman ProbeProbe ModificationsModifications

Increase thermal duplex stability DNA LNA Improve specificity Base Base O O Raise Tm by up to 8°C per LNA O O

Allow shorter probe design (~13bp) O O O

- (www.proligo.com) O P O O P O-

2'-O, 4'-C methylene bridge locks conformation IntroductionIntroduction toto MxProMxPro softwaresoftware

Critical Setting

•Threshold •Baseline AnalysisAnalysis termterm settingssettings AlgorithmsAlgorithms

• Developed using real Q-PCR training data, establish settings and ranges • Performs optimally for the majority of the fluorescence signal analyzed • Allows a user to analyze the raw data using the same method over time, identify trends • Easier to justify settings for validation, QA/QC purposes ThresholdThreshold ValueValue

Separates the data from the noise. Valid for Ct calculations if placed during exponential amplification.

3 Options: – Amplification based threshold (Minimizes variability between replicates) – 10 times the noise during early cycles. – Manual (click and drag) • On exponential phase. • Lines are parallel. • Minimize variability ThresholdThreshold-- AmplificationAmplification BasedBased BaselineBaseline SubtractionSubtraction

35000

30000 R

25000 R 20000 1

R 15000 dR

10000 dR 5000 1

0 010203040 -5000 Cycle # AssayAssay DevelopmentDevelopment andand ValidationValidation

Xn=X0 (1+E)n QQ--PCRPCR AssayAssay DesignDesign ConsiderationsConsiderations

• Consistency, Consistency, Consistency • Initial optimization efforts should identify good control or standard materials that you can rely upon throughout data generation • Generate a range of acceptable Q-PCR performance data • Controls should dictate what data is good or bad QQ--PCRPCR AssayAssay ProcessProcess

Experimental Design Design Oligo Design

Synthesis

Test oligos Gel Primers

Optimization Probe Enzyme, dNTP, Mg++ Validation

Run and Analyze ExperimentalExperimental DesignDesign Replicates n Depends on biological Biological variability (CV/Power Analysis)

Reflects experimental Technical(qPCR) error (n=3 is sufficient)

Independent Ensures biological relevance experiments (n=2 is sufficient)

Concordance of Results? GeneralGeneral StrategyStrategy forfor NewNew QPCRQPCR AssayAssay DevelopmentDevelopment • Plan to optimize assay using SYBR Green chemistry – SYBR melt curve will yield PCR specificity info that probe based detection will not – Attempt to constrain assays to a common thermal profile for convenience • Design amplicons compatible with probe chemistry for possible future use in a multiplex QPCR format ComponentsComponents ofof aa QualityQuality QPCRQPCR AssayAssay • QPCR amplification (Ct Linearity & Reproducibility, Efficiency, Sensitivity) – Standard curve should be run during assay optimization – High efficiency correlates with sensitivity of detection – Establishes a measuring range of assay • PCR amplification specificity from dissociation curve (Tm) FirstFirst AssayAssay -- TestingTesting OligosOligos

• First assay should be a standard curve run to test primers and overall assay performance – Dilution series, (1:5) X 6 points in triplicate, negative controls – 150 to 300nM primers, ~100 ngs of template (25nM to 1000nM) (25ngs to 250ngs) AssayAssay ValidationValidation Standard Curve Metrics Serial dilution of a positive control or amplified target Standard Curve

%Eff =10[-1/slope] -1

Log transform Ct Ct

Quantity LogQuantity Expect: High efficiency (E = 90 – 110%) Good linear fit (R2 > 0.98) 3-5 logs of dynamic range ~1 % CV variation among triplicates AssayAssay ValidationValidation Standard Curve Metrics

Eff=105.6% R2=0.97 AssayAssay ValidationValidation Standard Curve Metrics Select the best performing and most appropriate range for samples to be run

Copies

Eff=105.0% R2=1.0 11.5-30.2 Cts PrimerPrimer SelectionSelection

• Try to achieve similar Tm for all primers: Ideal ~60°C. (Future multiplexing or use of Taqman™ assays in mind)

• Forward and reverse primer should have ∆Tm <2°C (SYBR: 75 – 400, 200bp ; Taqman 75-150, 125bp)

• 40-60% GC content to prevent G/C region self-hybridization

• ∆G of primer dimer/cross primer dimer formation > -4 kcal/mol to avoid stable primer dimers

• Design via software (Always use the same one):

• Always perform a BLAST search with your amplicon and primers (Æ Specificity of the PCR) AssayAssay OptimizationOptimization PrimersPrimers WhatWhat ifif II cancan notnot identifyidentify primersprimers sequencessequences inin mymy genegene ofof interestinterest thatthat havehave thethe samesame annealingannealing temp?temp?

Tm of primers depends on concentration: perform a primer matrix test to identify optimal concentration using SYBR chemistry

SYBR based 50 nM 100 nM 150 nM 300 nM 600 nM 50 nM

100 nM

150 nM

300 nM

600 nM AssayAssay OptimizationOptimization PrimersPrimers Primer titration 50 nM – 200 nM duplicates for pos. Control & NTC Aims: Â low Ct values Æ sensitivity

100/150  no unspecific amplification or 150/100 positive primer dimers 150/100 controls Æ specificity NTC ∆Ct = 3 NTCs  Low interreplicate 100/150 variability NTC

 high efficiency of amplification QPCRQPCR AssayAssay ControlsControls Initial efforts should identify good control materials to run during assay setup and validation – Establish a range of acceptable QPCR performance data – Controls will dictate what data is good or bad and what should be included in down- stream analysis. – Justification for omitting data or re-assay QPCRQPCR ASSAYASSAY CONTROLSCONTROLS

Review the most common controls to include in any QPCR experiment – Systematic Experimental Error Control – Positive QPCR controls – Negative QPCR controls QPCRQPCR AssayAssay ControlsControls PassivePassive ReferenceReference FluorFluor Passive Reference Fluor (ROX) spiked into QPCR master mix at outset of assay setup – Rox fluor emission used to correct for artifacts in signal measurement from wells • Bubbles in sample volumes, plasticware inconsistency, variation in sample volume. – Include Rox, measure signal, assign it as the Reference Dye in Mx software setup – Will improve data uniformity and reduce correlation of variance (%CV) among technical replicates QPCRQPCR AssayAssay ControlsControls PassivePassive ReferenceReference FluorFluor-- ExampleExample Signal uniformity across 96 replicate wells QPCRQPCR AssayAssay ControlsControls PassivePassive ReferenceReference FluorFluor-- ExampleExample ~8% CV of Raw (R) Rox Signal across 96 wells QPCRQPCR AssayAssay ControlsControls PassivePassive ReferenceReference FluorFluor-- ExampleExample ~8% CV of Raw (R) Fam Signal across 96 wells QPCRQPCR AssayAssay ControlsControls PassivePassive ReferenceReference FluorFluor-- ExampleExample <1% CV of Ct for Rox normalized, baseline subtracted (dRn) across 96 wells QPCRQPCR AssayAssay ControlsControls PositivePositive QPCRQPCR ControlControl

Positive Controls- Common Sources of material – Pooled RNA/cDNA unknowns from experiments – Linearized/nicked plasmid cDNA – Purified PCR product – Stratagene Reference RNAs QPCRQPCR AssayAssay ControlsControls PositivePositive QPCRQPCR ControlControl

Positive Controls- Some sample that contains your gene of interest (GOI) and should be detected by QPCR – Ideal control should be similar to the unknowns you will be analyzing, ie RNA in same matrix as tissue or cell samples QPCRQPCR AssayAssay ControlsControls NegativeNegative QPCRQPCR ControlsControls • No Template controls (NTC) – No cDNA added to QPCR reaction – Detects primer dimer, contaminating template, or probe degradation across cycles • No Reverse Transcription Control (NoRT) – RNA sample undergoing reaction w/o RT – Detects contaminating gDNA in RNA • No Amplification Control (NAC) – No Taq DNA polymerase added to QPCR reaction – May indicate high background QPCRQPCR AssayAssay ControlControl SpecificitySpecificity NegativeNegative QPCRQPCR ControlControl Deriviation of fluorescence (R’T) Melting Temp. (Tm) Standard QPCRQPCR AssayAssay ControlControl SpecificitySpecificity NegativeNegative QPCRQPCR ControlControl

Sample

NTC, NoRT QPCRQPCR AssayAssay ControlControl SpecificitySpecificity NegativeNegative QPCRQPCR ControlControl

Standard QPCRQPCR AssayAssay ControlControl SpecificitySpecificity NegativeNegative QPCRQPCR ControlControl

Primer dimers BAD ! NTC QPCRQPCR AssayAssay ControlControl SpecificitySpecificity NegativeNegative QPCRQPCR ControlControl

Standard QPCRQPCR AssayAssay ControlControl SpecificitySpecificity NegativeNegative QPCRQPCR ControlControl

BAD ! gDNA Primer NoRT dimers NTC QPCRQPCR AssayAssay ControlControl SummarySummary

• Assay controls are the main determinant of data quality • Provide leverage for troubleshooting, allows you to regain assay performance quickly • Easy to prepare, requires up-front effort, worth the work in the long term QPCR Listserver – [email protected]

Contact Stratagene Technical Services – (800) 894-1304, Pacific Standard Time – [email protected]

Webinars and Introduction to QPCR Guide: – www.stratagene.com/fasttrack – An Introduction to Stratagene's Mx QPCR Software – Principle of Quantification by Real-Time PCR – Assay Validation and Optimization – Basic Assay Troubleshooting – QPCR Assay Controls – Critical Components of Assay Design – Enhancements offered in Stratagene’s MxPro™ QPCR Software ComparativeComparative QuantificationQuantification Given two samples: What is the difference in gene expression? Control Unknown

∆CtGOI Gene of Interest Ct Ct [Ct -Ct ] (1+E) C U

÷

∆CtNorm Normalizer Ct Ct [Ct -Ct ] (1+E) C U

Norm. ratio Thank You

Dan Schoeffner, Ph.D Field Applications Scientist [email protected] Tech. Services 800-894-1304

Lisa Thompson Technical Sales Representative [email protected]