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

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

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

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

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

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

Problematic qPCR curves Aurita Menezes Integrated DNA Technologies

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Aurita Menezes Integrated DNA Technologies

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

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

Ideal Standard Curves 102% Aurita Menezes Integrated DNA Technologies

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

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

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

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