Troubleshooting qPCR: What Are My Amplification Curves Telling Me?

Integrated DNA Technologies

TROUBLESHOOTING qPCR:What Are My Amplification Curves Telling Me?

Aurita Menezes, PhDAurita Menezes, PhDqPCR Product Manager

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Overview

Basics of an 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

Basics of an Amplification Curve

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Phases of an Amplification Curve

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R, Rn and Delta Rn

R= Multicomponent view (fluorescence obtained without any normalization)

Rn: Normalized reporter signal = emission intensity of the reporter dyeemission intensity of the passive reference dye (ROX)

ΔRn = Rn – background fluorescence

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Linear Rn View Log Baselined dRn

Baseline stop value should be set 1 to 2 cycles before earliest amplification cycleBaseline should be set in the linear view

Improper Baseline

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Proper Baseline

Linear View Log View

Baseline stop value should be set 1 to 2 cycles before earliest amplification cycleBaseline should be set in the linear view

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Good Threshold – in exponential phase

Bad Threshold – in plateau phase

Bad Threshold – in baseline phase

Threshold

Linear Scale

Logarithmic Scale

Linear view for Baseline settingLog view for Threshold setting

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Commonly Observed Problematic qPCR curves

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No Amplification

Lack of 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

Good efficiency

Poor efficiency

PCR efficiency

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PCR Efficiency

Lower efficiency 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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Unexpected PCR Efficiency…..Incorrect Dilutions

114%

Template conc. too high

Incorrect dilutions

100%

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Delayed Cq

Decreased efficiency Sample inhibition Incorrect normalizer concentration Master mix differences

The shift due to 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 EfficiencyEffect of SNPs within primer locations on Tm

PrimeTime® Predesigned qPCR Assays for Human, Mouse, and Rat

• Designed to avoid SNPS• We share primer and probe sequences upon purchase

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Delayed Cq……Sample Inhibition

Sample inhibition 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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HPRT TBP

MasterMix A

MasterMix BMasterMix A

MasterMix B

10 fold dilutions

Delayed Cq……Master Mixes Can Make a Difference

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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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Delayed Cq……Lower Fluorescent Dye Intensity

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Early Cq…..Too Much Template

Too much template Cq value comes up before 15 True amplification is observed when analyzed in the linear view

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Early Cq…..Automatic Baseline Failure

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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Earlier than expected Cq

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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Scattered Replicates

Pipetting Errors 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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Scattered Replicates…..Low Copy Number

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Height of Amplification Curve

Lowered background Probe concentration Signal bleed over Incorrectly assigned detector Increased ROX in samples Master mix

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Lowered background due 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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Regular qPCR Dual-Labeled Probes

ZEN™ Double-Quenched Probes

Dyes FAM, TET, HEX™, MAX, or JOEInternal 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

Case Study—How ZEN™ DQP Makes the DifferenceAdding 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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Height of Amplification Curve……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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Height of Amplification Curve….Not Enough ROX

Noisy signal

10 nM ROX

50 nM ROX

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Height of Amplification cCurve….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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Height of Amplification Curve……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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Height of Amplification Curve…..Multiplexing Optimized

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Unexpected Signal…

Positive NTC, 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 40 45

Positive NTC

Negative NTC

Threshold line

True amplification in No template Control

Unusual Curves

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Unusual Curves……….Sample Evaporation

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Unusual Curve…..Complete Evaporation of Sample

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Unusual curve…………Too Much Probe (6X)

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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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Unusual Curves….Amplification Beyond Plateau

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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Unusual Curve…… Amplification Beyond Plateau

When ROX normalization is turned off, the curve looks normal

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Unusual curves…….Too Much Template

dRn

SYBR® Melt Curves

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Melt Curves, An Indicator, 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.

It Takes More Than 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.

qPCR Resources: Webinars & Technical Info

For More information please visit www.idtdna.com Support Tech & Ed. Materials

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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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Thank you

Questions ?