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 ?