Troubleshooting qPCR: What are my amplification curves telling me?

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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.

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  • 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
  • Troubleshooting qPCR: What are my amplification curves telling me?

    1. 1. Troubleshooting qPCR:What are my amplification curves telling me?Aurita Menezes, Ph.D., Scientific Applications SpecialistIntegrated DNA Technologies
    2. 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. 3. Basics of an Amplification Curve Background Aurita Menezes Integrated DNA Technologies
    4. 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. 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. 6. Improper Baseline and Threshold Linear Rn View Log Baselined ΔRn Aurita Menezes Integrated DNA Technologies
    7. 7. Problematic qPCR curves Aurita Menezes Integrated DNA Technologies
    8. 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. 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. 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
    11. 11. Unexpected PCR Efficiency…..Incorrect dilutions 114% Template conc. too high Incorrect dilutions 100% Aurita Menezes Integrated DNA Technologies
    12. 12. Delayed Cq Decreased efficiency Low expression Sample inhibition Incorrect normalizer concentration Master mix differences Aurita Menezes Integrated DNA Technologies
    13. 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. 14. Delayed Cq……Lower fluorescent dye intensity combined with suboptimalEfficiency issues instrument optics for Dye B Dye A Dye B Aurita Menezes Integrated DNA Technologies
    15. 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. 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. 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. 18. Early Cq…..Automatic baseline failureWhen too much template is present, it’s likely that the instrument’ssoftware is unable to distinguish between noise and true amplification.In such cases, auto baseline may assign an incorrect value for thebaseline correction factor.  Adjust the baseline manually to correct this problem Aurita Menezes Integrated DNA Technologies
    19. 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 baselineReplicates ideally should not bemore than 0.5 Cq apart Aurita Menezes Integrated DNA Technologies
    20. 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. 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. 22. Height of Amplification Curve……Incorrect probe concentration Correct Probe Concentration Incorrect Probe Concentration Aurita Menezes Integrated DNA Technologies
    23. 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. 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
    25. 25. Unusual curves……….Sample evaporation Aurita Menezes Integrated DNA Technologies
    26. 26. Unusual curves…………TooNoisy signal- too much probe much probe (6X) Aurita Menezes Integrated DNA Technologies
    27. 27. Unusual curve…….Negative curves Dye calibration issues on instrument Aurita Menezes Integrated DNA Technologies
    28. 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. 29. Unusual curves….Amplification beyond plateau Aurita Menezes Integrated DNA Technologies
    30. 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. 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-synthesishttp://www.idtdna.com/pages/products/genes/gblocks-gene-fragmentshttp://www.idtdna.com/pages/products/dna-rna/ultramer-oligos Aurita Menezes Integrated DNA Technologies
    32. 32. Aurita MenezesIntegrated DNA Technologies
    33. 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. 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
    35. 35. Ideal Standard Curves 102% Aurita Menezes Integrated DNA Technologies
    36. 36. Height of Amplification Curve….. Level of ROX Least ROX ROX Normalization =OFF High Rox ROX Normalization=ON Aurita Menezes Integrated DNA Technologies
    37. 37. Unusual Curve…..Complete evaporation of sample Aurita Menezes Integrated DNA Technologies
    38. 38. Scattered Replicates...Low copy number Aurita Menezes Integrated DNA Technologies
    39. 39. Delayed Cq…..High ROX in reaction Differences in ROX concentration Aurita Menezes Integrated DNA Technologies

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