Hi Everyone,
I am doing quantitative PCRs to estimate the expression of protein coding genes. I have two questions about estimating primer/PCR efficiencies directly from amplification (fluorescence) data, i.e., without having to do a dilution series.
Thank you!
Reference
1. Peirson, S. N., Butler, J. N. & Foster, R. G. Experimental validation of novel and conventional approaches to quantitative real‐time PCR data analysis. Nucleic Acids Res. 31, e73–e73 (2003).
1) Add 1. Some use N(c) = N(0) * (1+E)^c (with 0 < E < 1), others use N(c) = N(0) * E^c (with 1 < E < 2).
2) I generally advice against estimating an efficiency based on individual curves. There are only 3-4 cycles (at best) that provide information about the efficiency, what ist very unreliable. It's ok for a rough guess, to check if there is a particular sample with problems, so that it's Ct value should not be used. But this is it. And for this purpose, it really does not matter if you use Rn, deltaRn, R or deltaR. When using Rn or R, some more (possibly non-linear) background-correction might be neccesary. (Note: the "delta" is for the difference in signals before and after the amplification cycle; the "n" is for fluorescence normalized to a passive reference dye. The "delta" is relevant mainly for TaqMan assays where reporter fluorescence from previous cycles accumulates).
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