I recently performed a qPCR for three different cell population and the ct values for 18S differed quite a lot (e.g. something like ~19, ~22 and ~25). A colleague insists it is my fault (pipetting error) but I just have a hard time believing so for several reasons. Can anyone provide their opinion on this?
One other question, let's say I performed a qPCR on three different cell populations (A = GFP-, B = GFP low, and C = GFP hi) and used 18S as the endogenous control and GFP is my target gene. To analyze my results, am I right to think that I should calibrate the machine this way:
Population A GFP ---> Population A, B, C 18S and GFP samples (basically using one population control for all the samples).
Or should I be using Population A 18S as the calibrator for every sample?
I'm using the Applied Biosystems 7500 Real-Time PCR System if it matters.
Edited by Wek, 14 July 2013 - 06:20 PM.