1. Troubleshooting qPCR: What are my amplification curves telling me?
Aurita Menezes, Ph.D., Scientific Applications Specialist
Thank you Hance. As Hance mentioned,in my role as the Scientific Application specialist I am expected to support researchers in different aspects of troubleshooting. Often it is just the amplification curve that is provided and I then need to deduce what could possibly have gone wrong with an experiment. So this webinar is aimed to give you some troubleshooting clues as to what can possibly be the issue based on the shape of the amplification curve

2. Overview Basics of an Amplification Curve Problematic qPCR Curves
Phases of an amplification curve
Terminology
Setting the correct baseline and threshold
Problematic qPCR Curves
No amplification
Unexpected efficiency
Delayed and early Cq
Scattered replicates
Unusual curves
Noisy signal
Amplification beyond plateau
Negative curves
Before I discuss various problematic qPCR curves, I am first going to cover the basic phases of an amplification cuve, and give an explanation of some of the commonly used terminology such as R, Delta R etc. We will also discuss the importance of setting the correct baseline and threshold . Finally we will cover troubleshooting various qPCR amplification curves
Aurita Menezes
Integrated DNA Technologies

3. Basics of an Amplification Curve
Background
The 3 main phases of an amplification curve are described in this picture. The baseline region is the time that amplification is occurring , however the amount of fluorescence does not rise above the level of background due to the limitations in sensitivity of the detector and lack of significant accumulation of amplicon
In the second phase, fluoresence is observed consistent with exponential amplification.
Although not depicted in this picture the next phase is the linear phase wherein reagents are being utilized and amplification is no longer exponential but does continue in a linear fashion before it plateaus.
Aurita Menezes
Integrated DNA Technologies

4. R, ΔR, Rn, and ΔRn R= Multicomponent view
(fluorescence obtained without any normalization)
∆R= Fluorescence - Baseline
Rn: Normalized reporter signal=emission of the reporter dye
emission of the passive reference dye (ROX)
ΔRn = Rn – baseline fluorescence
There is a lot of different ways one can view amp curves.
Commonly the X axis is defiined by the cycle number.
The Y axis can have the fluorescence data expressed in different ways
R is the raw fluorescence data obtained from all the channels that were selected on an instrument. So typically if one has a Fam probe with a Rox mastermix, one would expect to see the fluorecence due to Fam increase with amplification and Rox fluorescence to be a detectable signal but a straight line. If no other dye was used such as in a multiplex, all other channels should show no fluorescnece detected.
Thie R view is most useful in troubleshooting issues such as calibration as it tells you the amount of fluoreecence observed by the instrument without any number cruching
Delta R is the same data , but baseline s removed ,
Rn is the view wherein the raw fluorecence is expressed as a ratio that ha s been normalized to a reference dye such as Rox, so here the background is not removed ,
Finally delta Rn is where the baseline has been removed ( notice the y axis is 0) as well as the fluorescence is normalized
Aurita Menezes
Integrated DNA Technologies

5. Baseline and Threshold
Linear View
Log View
Baseline stop value should be set 1 to 2 cycles before earliest amplification
Set Baseline in Linear View
Set Threshold in Log View
Setting the correct baseline is important as it determines how much fluoresnece will be subtracted by the software in determining delta Rn.
On the left we have the same amp curve in the linear view and on the right hand side, it is set in the log view. Baseline should be set in the linear view and 1 to 2 cycles before amp take s off as you don’t want to subtract more signal than needed.
Most softwares automatically set threshold, but typically it is easier to set it in the log view as the exponential phase is best visualized
Aurita Menezes
Integrated DNA Technologies

6. Improper Baseline and Threshold
Linear Rn View Log Baselined ΔRn
Here we have an example of an improper baseline as well as threshold. As you can see if baseline is set after amplification is observed in the linear view, the curve is affected as it eliminates part of the amp curve when observed in the log view
Aurita Menezes
Integrated DNA Technologies

7. Problematic qPCR curves
Aurita Menezes
Integrated DNA Technologies

8. No Amplification No amplification Incorrectly assigned dye detector
Make sure instrument setting for dye
matches dye used in probe
Missing a master mix component
Repeat the experiment
Sample degradation
Does a different cDNA prep give
you the same result?
Lack of target in sample
Test a positive control
Assay design
Try a different assay
Machine not calibrated for dye
Calibrate the instrument
FAM incorrectly assigned as TAMRA
FAM incorrectly assigned as TET
The most common qPCR issue is the lack of amplification for which there can be many reasons as listed . If no amplification is observed, I would first check if no mistakes were made in selecting the right detector.
Here we have two examples, on the top we have the FAM incorrectly assigned as TAMRA and the bottom it has been incorrectly assigned as TET.
Secondly I would repeat the experiment to make sure that one did not forget to add a component during set up as well as try and repeat the experiment with a different template or CDNA prep. Finally it is quite likely that the target is simply not expressed in your sample , so a positive control is absolutely essential , as if one does have a positive control such as a plasmid gene, it helps determine if the problem was the assay or the sample
Aurita Menezes
Integrated DNA Technologies

9. Unexpected PCR Efficiency
Lower efficiency (<85%)
Incorrect dilutions causing errors in standard curve
Not enough dynamic range of standard curve
Primers designed on a SNP site
Lower fluorescence of dye
Instrument not calibrated for dye
Sample inhibition
Higher efficiency (>110%)
Genomic DNA contamination
Incomplete DNase treatment
Another frequent issue is that the pcr efficiency is not as expected. Some of the reasons are listed here,
however the most frequent issue that can contribute to both high and low efficiency is errors in dilutions and not having a dynamic range in the generation of a standard curve
Aurita Menezes
Integrated DNA Technologies

10. PCR Efficiency Efficiency reflects whether DNA doubled every cycle
It takes 3.32 cycles for DNA to be amplified 10 fold
If samples have been correctly diluted, every 10-fold dilution should be 3.32 cycles
During PCR if DNA doubles every cycle then, it Is considered to be 100% efficient.
So ideally what this means is that for a DNA template to get amplified 10 fold, it takes 3.32 cycles. So if you are making 10 fold dilutions in the generation of the standard curve, one would expect that the amplification curves would be 3.32 cycles apart
An ideal standard curve will also have all its replicates within 0.5 Cq of each other and i your R2 value reflects how your dilutions and replicates fit on your standard curve. Ideally you should get standard curves with R2 value of 0.99
Aurita Menezes
Integrated DNA Technologies

11. Unexpected PCR Efficiency…..Incorrect dilutions
114%
Template conc. too high
Incorrect
dilutions
100%
Aurita Menezes
Integrated DNA Technologies

12. Delayed Cq Decreased efficiency Low expression Sample inhibition
Incorrect normalizer concentration
Master mix differences
In the next few slides we are going to discuss these reason for a delayed Cq
Aurita Menezes
Integrated DNA Technologies

13. Delayed Cq……..Lower efficiency
If 10-fold dilutions are all >3.32 cycles apart:
Are your primers on a SNP site?
Consider using IDT PrimeTime® Predesigned Assaysdesigned to avoid SNP sites through the use of updated sequence information from NCBI databases
Here is an example of a standard curve wherein all the 10 fold dilutions are greater than 3.32 cycles apart. This equally distributed delayed Cq could be due to suboptimal primer design.
Aurita Menezes
Integrated DNA Technologies

14. Delayed Cq……Lower fluorescent dye intensity combined with suboptimal
Delayed Cq……Lower fluorescent dye intensity combined with suboptimal instrument optics for Dye B
Efficiency issues
Dye A
Dye B
Aurita Menezes
Integrated DNA Technologies

15. Delayed Cq……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 contamination with phenol layer during RNA isolation
10-fold dilution
In this example the first dilution appears to be more deviant from the rest of the samples on the standard curve
Aurita Menezes
Integrated DNA Technologies

16. Delayed Cq……Master mixes can make a difference
Master Mix A
Master Mix A
Master Mix B
Master Mix B
10-fold dilutions
HPRT TBP
Aurita Menezes
Integrated DNA Technologies

17. Early Cq…..Too much template
Cq value comes up before cycle 15
True amplification is observed when analyzed in the linear view
Aurita Menezes
Integrated DNA Technologies

18. Early Cq…..Automatic baseline failure
When too much template is present, it’s likely that the instrument’s software is unable to distinguish between noise and true amplification. In such cases, auto baseline may assign an incorrect value for the baseline correction factor.
Adjust the baseline manually to correct this problem
Aurita Menezes
Integrated DNA Technologies

19. Scattered Replicates Pipetting errors
Poor thermal calibration (thermocycler is raising and lowering temperature inconsistently across different wells)
Denaturation time is too short (if using a fast cycling master mix, consider increasing denaturation time from 5 to 20 sec.)
Low copy number
Incorrectly set baseline
Replicates ideally should not be
more than 0.5 Cq apart
Aurita Menezes
Integrated DNA Technologies

20. Height of Amplification Curve
Lowered background
Probe concentration
Signal bleed over
Incorrectly assigned detector
Increased ROX in samples
Master mix
Aurita Menezes
Integrated DNA Technologies

21. Height of Amplification Curve…..
Lowered background due to improved quenching
IDT double-quenched ZEN™ probes (available with IDT PrimeTime® qPCR Assays) have lower background and increased sensitivity
Aurita Menezes
Integrated DNA Technologies

22. Height of Amplification Curve……Incorrect probe concentration
Aurita Menezes
Integrated DNA Technologies 22

23. Height of Amplification Curve…. Amount of ROX
50 nM ROX
100 nM ROX
Noisy signal
10 nM ROX
50 nM ROX
Aurita Menezes
Integrated DNA Technologies

24. Height of Amplification Curve……Multiplex vs. Singleplex
The height of amplification curve is typically lowered when a target is investigated in a multiplex reaction vs. a singleplex reaction.
More importantly, it is critical that the Cq is not shifted between both reactions.
If multiplexing,
The master mix needs to be
adjusted for additional
dNTPs, Mg, and Taq enzyme or
Use a master mix
specifically designed for
multiplexing
Singleplex
Multiplex
Aurita Menezes
Integrated DNA Technologies

25. Unusual curves……….Sample evaporation
Aurita Menezes
Integrated DNA Technologies

26. Unusual curves…………Too much probe (6X)
Noisy signal- too much probe
Aurita Menezes
Integrated DNA Technologies

27. Unusual curve…….Negative curves
Dye calibration issues on instrument
Aurita Menezes
Integrated DNA Technologies

28. Unusual curve……..Negative curves
If the instrument is not correctly calibrated,
Fluorescence due to amplification increases in a given channel, however the fluorescence attributed to background will also increase, while fluorescence attributed to the other dyes and the normalizer may be artificially lowered resulting in negative curves
Calibrate the machine
again for all the dyes
being used
Aurita Menezes
Integrated DNA Technologies

29. Unusual curves….Amplification beyond plateau
Aurita Menezes
Integrated DNA Technologies

30. Unusual curves…. Amplification is observed beyond plateau
When ROX normalization is
turned off,
the curve looks normal
Amplification is observed beyond plateau
Fluorescence detected is at maximum capacity for the detector
Consequently, the amount of fluorescence attributed to Rox is mistakebly decreased as the amount of fluorescence attributed to back ground increases.
Consequently fluorescence is normalized to a smaller Rox value, artificially increasing the heinght of the amp curve
Turn normalizer off
Aurita Menezes
Integrated DNA Technologies

31. Summary
Information on PrimeTime® qPCR Assays with ZEN™ double-quenched probes and PrimeTime® qPCR Primers can be found at:
For background on setting up qPCR experiments, qPCR protocols, and troubleshooting information like that presented in this webinar, download the IDT PrimeTime® qPCR Application Guide at:
Information on products that can be used as controls such as MiniGenes™, gBlocks™, Ultramers™ and can be found at:
Aurita Menezes
Integrated DNA Technologies

32. Aurita Menezes
Integrated DNA Technologies

33. Unexpected Signal…
Positive NTC -> maybe master mix got contaminated with template during qPCR prep
Positive –RT -> gDNA contamination
Incomplete DNase treatment
Assay design
Aurita Menezes
Integrated DNA Technologies

34. Threshold Good Threshold – in exponential phase Bad Threshold –
in plateau phase
in baseline phase
Linear Scale
Logarithmic Scale
Aurita Menezes
Integrated DNA Technologies

35. Ideal Standard Curves
102%
Aurita Menezes
Integrated DNA Technologies

36. Height of Amplification Curve….. Level of ROX
ROX Normalization =OFF
Least ROX
ROX Normalization=ON
High Rox
Aurita Menezes
Integrated DNA Technologies

37. Unusual Curve…..Complete evaporation of sample
Aurita Menezes
Integrated DNA Technologies

38. Scattered Replicates...Low copy number
Aurita Menezes
Integrated DNA Technologies

39. Delayed Cq…..High ROX in reaction
Differences in ROX concentration
Aurita Menezes
Integrated DNA Technologies