Quantitative PCR (qPCR) is the method of choice for accurate estimation of gene expression. Part of its appeal for researchers comes from having a protocol that is easy to execute. However when your reactions do not result in ideal amplification, troubleshooting "why" can be challenging. Factors including sample quality, template quantity, master mix differences, assay design, and incorrect primer or probe resuspension can all influence efficient amplification. When troubleshooting, analysis of the appearance of your amplification curve can give you clues towards improving your results.
See the 2,456 pharmacies on the National E-Pharmacy Platform
Troubleshooting qPCR: What are my amplification curves telling me?
1. Troubleshooting qPCR:
What are my amplification curves telling me?
Aurita Menezes, Ph.D., Scientific Applications Specialist
Integrated DNA Technologies
2. Overview
Basics of an Amplification Curve
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
Aurita Menezes
Integrated DNA Technologies
3. Basics of an Amplification Curve
Background
Aurita Menezes
Integrated DNA Technologies
4. R, ΔR, Rn, and ΔRn
R= Multicomponent view
(fluorescence obtained Baseline any normalization)
∆R= Fluorescence - without
Rn: ΔRn = Rn – baseline fluorescence
Normalized reporter signal=emission of the reporter dye
emission of the passive reference dye (ROX)
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
Aurita Menezes
Integrated DNA Technologies
6. Improper Baseline and Threshold
Linear Rn View Log Baselined ΔRn
Aurita Menezes
Integrated DNA Technologies
8. NoNo Amplification
amplification
Incorrectly assigned dye detector
Make sure instrument setting for dye FAM incorrectly assigned as TAMRA
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
FAM incorrectly assigned as TET
Test a positive control
Assay design
Try a different assay
Machine not calibrated for dye
Calibrate the instrument
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%)
Incorrect dilutions causing errors in standard curve
Not enough dynamic range of standard curve
Genomic DNA contamination
Incomplete DNase treatment
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
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
Aurita Menezes
Integrated DNA Technologies
14. Delayed Cq……Lower fluorescent dye intensity combined with suboptimal
Efficiency issues
instrument optics for Dye B
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
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
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
Correct Probe
Concentration
Incorrect Probe
Concentration
Aurita Menezes
Integrated DNA Technologies
23. Height of Amplification Curve…. Amount of ROX
50 nM ROX
50 nM ROX
100 nM ROX
Noisy signal
10 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 Singleplex
or Multiplex
Use a master mix
specifically designed for
multiplexing
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
30. Unusual curves….
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
When ROX normalization is
back ground increases.
turned off,
Consequentlycurve looks normal normalized to a smaller Rox value,
the fluorescence is
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:
http://www.idtdna.com/pages/products/gene-expression/primetime-qpcr
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:
http://www.idtdna.com/pages/support/technical-vault/reading-room/user-guides-
protocols
Information on products that can be used as controls such as MiniGenes™,
gBlocks™, Ultramers™ and can be found at:
http://www.idtdna.com/pages/products/genes/custom-gene-synthesis
http://www.idtdna.com/pages/products/genes/gblocks-gene-fragments
http://www.idtdna.com/pages/products/dna-rna/ultramer-oligos
Aurita Menezes
Integrated DNA Technologies
34. Threshold
Linear Scale
Logarithmic Scale
Bad Threshold – Good Threshold – Bad Threshold –
in plateau phase in exponential phase in baseline phase
Aurita Menezes
Integrated DNA Technologies
36. Height of Amplification Curve….. Level of ROX
Least ROX ROX Normalization =OFF
High Rox
ROX Normalization=ON
Aurita Menezes
Integrated DNA Technologies
39. Delayed Cq…..High ROX in reaction
Differences in ROX concentration
Aurita Menezes
Integrated DNA Technologies
Editor's Notes
Thank you Hance. AsHancementioned,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
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
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.
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 waysR 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 Roxmastermix, 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 cruchingDelta 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
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
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
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
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
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 apartAn 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
In the next few slides we are going to discuss these reason for a delayed Cq
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.
In this example the first dilution appears to be more deviant from the rest of the samples on the standard curve