What is the best excel-based simplified way or freely-available sofrware-based method for the calculation of RT-qPCR effeciencies in relative quantification of gene expression?. I normally use a standard curve method with 10-fold cDNA dilution series and from the slope I use the formula E = 10 exp.-(1/slope) . Any easier ways implementing, for example, macros in excel interface?.
If you are using the Applied biosystems7900Fast Real-Time PCR system, the software is SDSv2.2.1 or higher. However you need to export the files into excel before doing any data manipulation. I have not yet encountered a PCR system that interfaces directly with excel, and therefore some form of transfer is always necessary, but once its done things are not as complicated as they seem if the excel formulas are properly applied.
What can be simpler than a linear regression of ct versus log(N) ?
I recommend to use 1:2 dilution series. This is easier and more precisely to pipet. You also get a denser spacing, allowing a better judgement of the linear range. There is no need for technical replicated a one particular concentration. It is preferrable to have a different concentration in each PCR (example: instead of 1:2, 1:20, 1:200, each measured in triplicates, it is better to measure 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256. Both require 9 PCRs, but the latter gives you more information about the linearity; the standard error of the slope is not affected).
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