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Control inconsistence in Q-PCR - (Jun/01/2011 )


Im doing a Comparative Ct study with sybr green (takara) in a step One termocycler. I have eight target genes, 5 samples (4 treatment and one untreated control that is the reference sample, but I will add more samples in the future but not more genes) and i`m using RNA18S as an endogenous control. I use one 48 well plate for each different sample and I check all 8 genes in each one of the plates (I've just use one plate per sample because is more confortable to work, and because i saw that applied biosystems just did the same in the comparative Ct study example included in the software). I work doing 5 replicates, and I obtain not great Ct differences between genes duplicates in the same plate (0.1-0.4).

Now the question: I have just tried to run two different plates of the same sample (untreated control) and when I compare the genes I obtain 5-fold differences in all genes. What's happening?

RNA18S Ct is around 12-13 and the target genes Ct is around 23-27, this could be a problem? should I try to restart my experiments putting all my samples together?



please some feedback...