In regards to calibrators for relative fluorescence using the delta delta Ct method, what can (should) be used? Must it only be a certain treatment, or can it be a sample?
For example, say I'm analyzing the effect pH and temperature have on the expression of a group of bacterial genes, and I'm using 16s rRNA as the endogenous gene. The genes will be genA, genB, genC, and genD. The growth conditions would be ph 7 at 37 C, pH 5 at 37 C, pH 7 at 28 C, and pH 5 at 28 C. I want to calibrate based on the lowest expression.
Does the calibrator then have to be a temp/pH combination or can it be genA (or B or C or D)?
My understanding is that the calibrator must be a particular treatment (such as pH 7, 37 C), and you'd compare the delta Ct of the genes in the other treatments to the respective delta Ct values of the calibrator (pH 7, 37 C). However, I'm asking because I've been told by someone doing similar analysis that it's common to take the value of the weakest signal and use that as the calibrator. So, if genA was the weakest signal in each reaction, that would be used as the calibrator.
I've personally never seen relative qPCR calculated that way (it's always been with a treatment - or "untreated" - as being the calibrator), and am wondering if anyone has seen it calculated any way other than that?
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