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Different efficiency in different samples but the same primer - (Sep/17/2008 )

Hi, all,

I encounter a weired problem. My experiment condition is Nontreated, DMSO control, and Drug treated for 3, 5, and 7 days (The drug has to be dissolved first in DMSO). I did ChIP and RNA extraction on these samples and then quantitated them using Bio-Rad real-time PCR system to compare the effect of the different conditions on one gene (one primer set). For cDNA, I use B actin as internal control.

However, in all my experiments, the efficiency of DMSO control for day 3, 5, and 7 is always lower than the other condition (so the slope of its standard curve is <-3.5 and the manual says it should be around -2.2~-3.3) and this causes its quantitation results are higher than it should be (sometimes higher than Drug treated one). So I don't know whether I can trust the results or not. I think DMSO must have some effect on the template but why? Can I still compare my samples when the efficency of some samples is too low? Any suggestion for solving the problem? Thank you so much.



In normal PCR, DMSO is often added to efficiently amplify GC rich regions -- I believe it changes the melting temperature of the DNA, and its effect is concentration dependent. Knowing this, I would be surprised if you *didn't* see a difference between reactions run with DMSO and without, and I'd be surprised if you didn't see a difference between two reactions run both with DMSO but at different final concentrations.