1. Integrated DNA Technologies
TROUBLESHOOTING qPCR:
What Are My Amplification Curves Telling Me?
Aurita Menezes, PhD
Aurita Menezes, PhD
qPCR Product Manager

2. 2
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

3. Basics of an Amplification Curve

4. 4
Phases of an Amplification Curve
4

5. 5
R, Rn and Delta 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

6. 6
Linear Rn View Log 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

7. 7
Proper Baseline
Linear View Log View
Baseline stop value should be set 1 to 2 cycles before earliest amplification cycle
Baseline should be set in the linear view

8. 8
Good Threshold –
in exponential 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

9. Commonly Observed Problematic qPCR curves

10. 10
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

11. Good efficiency
Poor efficiency
PCR efficiency

12. 12
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

13. 13
Unexpected PCR Efficiency…..Incorrect Dilutions
114%
Template conc. too high
Incorrect
dilutions
100%

14. 14
Delayed Cq
 Decreased efficiency
 Sample inhibition
 Incorrect normalizer concentration
 Master mix differences

15. 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 Efficiency
Effect of SNPs within primer locations on Tm

16. PrimeTime® Predesigned qPCR Assays for Human, Mouse, and Rat
• Designed to avoid SNPS
• We share primer and probe sequences upon purchase

17. 18
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

18. 19
HPRT TBP
MasterMix A
MasterMix B
MasterMix A
MasterMix B
10 fold dilutions
Delayed Cq……Master Mixes Can Make a Difference

19. 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

20. 21
Delayed Cq……Lower Fluorescent Dye Intensity

21. 22
Early Cq…..Too Much Template
 Too much template
 Cq value comes up before 15
 True amplification is observed when analyzed in the linear view

22. 23
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

23. 24
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

24. 25
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

25. 26
Scattered Replicates…..Low Copy Number

26. 27
Height of Amplification Curve
 Lowered background
 Probe concentration
 Signal bleed over
 Incorrectly assigned detector
 Increased ROX in samples
 Master mix

27. 28
 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

28. 29
Regular qPCR Dual-Labeled Probes
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

29. 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.

30. 31
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

31. 32
Height of Amplification Curve….Not Enough ROX
Noisy signal
10 nM ROX
50 nM ROX

32. 33
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

33. 34
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

34. 35
Height of Amplification Curve…..Multiplexing Optimized

35. 36
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
Positive
NTC
Negative
NTC
Threshold line
True amplification in No template Control

36. Unusual Curves

37. 38
Unusual Curves……….Sample Evaporation

38. 39
Unusual Curve…..Complete Evaporation of Sample

39. 40
Unusual curve…………Too Much Probe (6X)

40. 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

41. 42
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.

42. 43
Unusual Curve…… Amplification Beyond Plateau
When ROX normalization is
turned off,
the curve looks normal

43. 44
Unusual curves…….Too Much Template
dRn

44. SYBR® Melt Curves

45. 46
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.

46. 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.

47. qPCR Resources: Webinars & Technical Info
For More information please visit www.idtdna.com  Support  Tech & Ed. Materials

48. 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

49. 50
Thank you
Questions ?