Which normalization method is better for relative comparative qPCR (ddCt): Endogenous control or Global Normalization?

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Milad Lagzian

ddct with endogenous control if its expression did not fluctuate at high level between your treatment and your control (dCt

Marcin Schmidt

Milad, I run several endogenous controls and selected the most stable one (it fits the dCt

Heidi, did you checked that fluctuations in efficiency is a matter of primers or maybe template/inhibitors? I compare several related treatments (of the same cell line) in relative comparative study and my interests are in changes of expression of the same genes between treatments. So different efficiencies are kind of a constant in my case (I assume). Correct me if I am wrong.

Heidi, I check efficiency at the optimalization stage and when I get an acceptable result consider it done, if not I try different primers. I never bothered myself with checking primers with standard curve during each run. I usually have full plate. However now I will check again.