Troubleshooting qPCR: What Are My Amplification Curves Telling Me?

Troubleshooting qPCR: What Are My Amplification Curves Telling Me?

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  Integrated DNA Technologies TROUBLESHOOTINGqPCR: What Are My Amplification Curves Telling Me? Aurita Menezes, PhD Aurita Menezes, PhD qPCR Product Manager
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  2 Overview  Basics ofan Amplification Curve  Terminology  Setting the correct baseline and threshold  Commonly Observed Problematic qPCR Curves  No amplification  Efficiency  Cq, delayed and early  Scattered replicates  Height of amplification curve  Unexpected signal in NTC  Unusual curves  SYBR® Melt Curves
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  Basics of anAmplification Curve
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  4 Phases of anAmplification Curve 4
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  5 R, Rn andDelta Rn R= Multicomponent view (fluorescence obtained without any normalization) Rn: Normalized reporter signal = emission intensity of the reporter dye emission intensity of the passive reference dye (ROX) ΔRn = Rn – background fluorescence 5
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  6 Linear Rn ViewLog Baselined dRn Baseline stop value should be set 1 to 2 cycles before earliest amplification cycle Baseline should be set in the linear view Improper Baseline 6
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  7 Proper Baseline Linear ViewLog View Baseline stop value should be set 1 to 2 cycles before earliest amplification cycle Baseline should be set in the linear view
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  8 Good Threshold – inexponential phase Bad Threshold – in plateau phase Bad Threshold – in baseline phase Threshold Linear Scale Logarithmic Scale Linear view for Baseline setting Log view for Threshold setting 8
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  Commonly Observed ProblematicqPCR curves
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  10 No Amplification  Lackof target in sample  Positive control  Assay design failure  Try a different assay  Sample degradation  Does a different cDNA prep give you the same result?  Machine not calibrated for dye being used  Calibrate the instrument  Incorrectly assigned dye detector  Make sure setting on instrument matches the probe being used Log Linear FAM assigned as TAMRA FAM assigned as TET
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  Good efficiency Poor efficiency PCRefficiency
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  12 PCR Efficiency  Lowerefficiency  Primers designed on a SNP site  Lower sensitivity of probe  Sample inhibition  Incorrect dilutions causing errors in standard curve  Higher efficiency (greater than 110%)  Primer dimers or nonspecific amplification  Incomplete DNase treatment  Incorrect dilutions causing errors in standard curve  Not enough dynamic range of standard curve
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  13 Unexpected PCR Efficiency…..IncorrectDilutions 114% Template conc. too high Incorrect dilutions 100%
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  14 Delayed Cq  Decreasedefficiency  Sample inhibition  Incorrect normalizer concentration  Master mix differences
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  The shift dueto a SNP at the 3′ end of a primer varies from 0 to >10 Cq’s. This shift misrepresents a gene expression fold change of as much 1000 fold Impact of SNPs on Primer Efficiency Effect of SNPs within primer locations on Tm
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  PrimeTime® Predesigned qPCRAssays for Human, Mouse, and Rat • Designed to avoid SNPS • We share primer and probe sequences upon purchase
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  18 Delayed Cq……Sample Inhibition Sampleinhibition  The concentration of inhibitors is maximum in the least dilute sample  As the sample is diluted, the inhibitory effect decreases  Make a new cDNA prep, try to minimize contaminantion with phenol layer during RNA isolation 10 fold dilution
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  19 HPRT TBP MasterMix A MasterMixB MasterMix A MasterMix B 10 fold dilutions Delayed Cq……Master Mixes Can Make a Difference
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  20 Delayed Cq……..Lower Efficiency If 10 fold dilutions are all greater than 3.32 cycles apart…  Are your primers on a SNP site?  Can a difference in primer Tms (> 5 °C) be producing unequal extension  Annealing temperature is too low  Unanticipated variants within the target sequence
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  21 Delayed Cq……Lower FluorescentDye Intensity
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  22 Early Cq…..Too MuchTemplate  Too much template  Cq value comes up before 15  True amplification is observed when analyzed in the linear view
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  23 Early Cq…..Automatic BaselineFailure  When too much template is present, it is likely that the software is unable to distinguish between noise and true amplification, thus auto baseline may incorrectly assign the value for the baseline correction factor  Adjusting baseline manually corrects this problem
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  24 Earlier than expectedCq  Genomic DNA contamination  Multiple products  High primer-dimer production  Poor primer specificity  Transcript naturally has high expression in samples of interest
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  25 Scattered Replicates  PipettingErrors  Poor thermal calibration (inconsistent raising and lowering of temperature across different wells in a thermocycler block)  Denaturation time is too short ( if using fast cycling master mix (consider increasing denaturation time from 5 to 20 secs)  Low copy number  Incorrectly set baseline
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  26 Scattered Replicates…..Low CopyNumber
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  27 Height of AmplificationCurve  Lowered background  Probe concentration  Signal bleed over  Incorrectly assigned detector  Increased ROX in samples  Master mix
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  28  Lowered backgrounddue to improved quenching  IDT ZEN™ Double-Quenched Probes (available with IDT PrimeTime® qPCR Assays) have lower background and increased sensitivity ZEN™ Double-Quenched Probes Height of Amplification probes…Lowered Background
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  29 Regular qPCR Dual-LabeledProbes ZEN™ Double-Quenched Probes Dyes FAM, TET, HEX™, MAX, or JOE Internal Quencher ZEN™ 3′ quencher Iowa Black® FQ FAM/ZEN/IaBlkFq is available as: • PrimeTime® Mini Probes—0.5 nmole delivered yield • PrimeTime® Eco Probes—2.5 nmole delivered yield Also available on starting synthesis scales of 100 nmole, 250 nmole and 1 µmole PrimeTime® qPCR—ZEN™ Double-Quenched Probes
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  Case Study—How ZEN™DQP Makes the Difference Adding a ZEN™ Internal Quencher decreases background fluorescence Figure 1A). Railing can lead to signal bleed over into adjacent channels, which can complicate data interpretation if those channels are also being used (Figure 1B). The reduced background fluorescence of ZEN™ Double-Quenched Probes compared to traditional single- quenched probes is demonstrated in Figure 1C.
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  31 Height of AmplificationCurve……Incorrect Probe Concentration Correct Probe Concentration Incorrect Probe Concentration Lowered height of amplification curve can also be due to limiting reagents or degraded reagents such as the dNTPs or master mix
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  32 Height of AmplificationCurve….Not Enough ROX Noisy signal 10 nM ROX 50 nM ROX
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  33 Height of AmplificationcCurve….Low ROX Normalized reporter signal (Rn): emission intensity of the reporter emission intensity of the passive reference dye (Rox) ΔRn = Rn – background noise 50 nM ROX 100 nM ROX
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  34 Height of AmplificationCurve……Multiplex vs Singleplex  Height of amplification curves are typically lowered when a target is investigated in a multiplex reaction in comparison to a singleplex reaction  More importantly though it is important that the Cq is not shifted between both reactions  If multiplexing, master mix needs to be adjusted for additional dNTPs, Mg2+ and Taq enzyme or use a master mix specifically designed for multiplexing Singleplex Multiplex
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  35 Height of AmplificationCurve…..Multiplexing Optimized
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  36 Unexpected Signal…  PositiveNTC, maybe master mix got contaminated with template during qPCR prep  Positive –RT = gDNA contamination  Incomplete DNase treatment  Assay design 0 5 10 15 20 25 30 35 Positive NTC Negative NTC Threshold line True amplification in No template Control
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  Unusual Curves
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  38 Unusual Curves……….Sample Evaporation
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  39 Unusual Curve…..Complete Evaporationof Sample
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  40 Unusual curve…………Too MuchProbe (6X)
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  41 Unusual Curve……..Negative Curves If the instrument is not correctly calibrated, when fluorescence due to amplification increases in a given channel, the fluorescence attributed to background increases, while fluorescence attributed to the other dyes may be decreased by the instrument  Calibrate the machine again for all the dyes being used
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  42 Unusual Curves….Amplification BeyondPlateau  Amplification is observed beyond plateau  Fluorescence detected is at maximum capacity for the detector  The amount of fluorescence attributed to ROX is mistakenly decreased as the amount of fluorescence attributed to back ground increases  Fluorescence is normalized to a smaller Rox value, artificially increasing the height of the amp curve  Turn normalizer off  Lower primer probe conc.
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  43 Unusual Curve…… AmplificationBeyond Plateau When ROX normalization is turned off, the curve looks normal
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  44 Unusual curves…….Too MuchTemplate dRn
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  SYBR® Melt Curves
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  46 Melt Curves, AnIndicator, Not a Diagnosis (A) An amplicon from CFTR exon 17b reveals a single peak following melt curve analysis, while (B) An amplicon from exon 7 produces 2 peaks, often considered as representing multiple amplicons.
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  It Takes MoreThan a Melt Curve C. uMelt Derivation Melt Curve for CFTR Exon 13. B. CFTR Exon 13 Agarose Gel. CFTR Exon 13 Melt Curve.  Shoulder peaks maybe due to low complexity regions in your amplicon that cause non-uniform melting  Typically, primer-dimers have a significantly lower melting temperature and present with a low, broad melting curve peak.
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  qPCR Resources: Webinars& Technical Info For More information please visit www.idtdna.com  Support  Tech & Ed. Materials
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  49 PrimeTime® qPCR Products Gene Expression Studies  Custom design in any species  ZEN™ Double-Quenched Probes  In human, mouse, and rat  PrimeTime® qPCR Predesigned qPCR Assay Database  Genotyping Studies  Custom design in any species  LNA PrimeTime® Probes and Mini LNA PrimeTime® Probes  Free Design Tools  Custom design in any species  PrimerQuest ® Tool, RealTime PCR Tool  Resources on the Web
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  50 Thank you Questions ?