I have just started doing qRTPCR using SYBR green (Roche) with ready made primers for the gene of interest (SABiosciences). I consistently find a reaction efficiency of greater than 100 in the range of 120-150 for all the genes I look at, including the housekeeping genes GAPDH and Actb, on a Bio-Rad CFX96 instrument. I prepare 10 fold serial dilutions from an untreated cell culture sample (BV2 microglia) and run a standard curve and use the (10^(-1/slope)-1)*100 formula to calculate reaction efficiency. The r2 value is in the range of 0.96-0.98.
I calculate the delta Ct values for all the genes by subtracting the Ct values of the gene of interest from the house keeping gene and the values are fairly constant (variation by 1 cycle at max) at all the concentrations measured. And this is the same whether I use GAPDH or Actb for subtraction (the absolute delta Ct values differ but they follow a similar pattern).
Can this negatively affect my results and the interpretation if I want to use the delta delta Ct method to measure relative changes in gene expression between different treatments for my actual experiment?
HI, your efficiency is too high with 120-150%! The accepted range for qPCR is
generally 80 to 110% (some paper say: 90 to 110%).
High efficiency: This might happen when you have PCR inhibitors present in your template. The inhibitors get diluted out with low copy numbers in the mixture, which can artificially raise your efficiency.
Another possibility is if you have non-specific products being amplified in the dilute samples that lower the Cts. You can check for this by running a gel with your PCR reaction products.
Cheers, Nadine
HI, your efficiency is too high with 120-150%! The accepted range for qPCR is
generally 80 to 110% (some paper say: 90 to 110%).
High efficiency: This might happen when you have PCR inhibitors present in your template. The inhibitors get diluted out with low copy numbers in the mixture, which can artificially raise your efficiency.
Another possibility is if you have non-specific products being amplified in the dilute samples that lower the Cts. You can check for this by running a gel with your PCR reaction products.
Cheers, Nadine
If you do measure an efficiency of more than 100%, then there is some obvious problem in the data (some among many, mostly unknown, possible reasons: wrong or suboptimal background correction, presence of PCR inhibitors, amplification of unspecific products, generation of eximers, ...). Given the errors are similar for all targets and all samples, then the interpretation of the delta-ct values as is still ok. What you should NOT do is to translate delta-delta-ct values into "(log) fold-changes". However, demonstrating a regulation or differential expression is possible. But I doubt that the errors are similar for all targets and samples... you write that your results vary between 120% and 150%, what is quite a large variation!
Two options:
Check the background correction and the presence of PCR inhibitors. If the problem lies here and you can fix it: fine. If not, go for an "absolute" quantification for each target. Then possible differences in efficiencies for the different targets are not relevant anymore.
Note: Efficiencies above 100% are an artifact. Although there are software available dealing with the problem of unequal efficiencies, I would not trust the results, because the reason for your efficiency estimate is clearly *not* that each dsDNA molecule is "more than doubled" during one cycle.
I just have an idea: if during the extension phase another primer binds ate the "breathing end" of a newly synthesized double strand and is extended and the old strand is displaced without being choped (Endo-minus polymerase), then a "more than doubeling" per cycle is at least thinkable (kind of partial isothermal amplification during the extension phase). This may be circumvented by shorter extension times and higher extension temperatures (use a three-step protocol) and primers with a lower Tm (so that the primers won't anneal at the extension temp). I think this scenario is quite unlikely... but one never knows...
I agree with Nadine, plus your r2 values are not very good. I havent seen your graphs but this could be reflecting PCR inhibition i.e. higher concentrations of cDNA (carrying more inhibitors) amplifying later than they should, compared to the more dilluted cDNA samples. What is the variation of the Cts among your samples (particularly the less concentrated ones?) Are they varying ~ 2.2 - 2.5 Cts, or less?
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