varying template concentrations

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dodsferd's picture
varying template concentrations

 Hello all,

I am currently using comparative qPCR (SybrGreen I) to compare the expression of a gene of interest in different samples (treated vs untreated, at different time-points), using an endogenous normalizer.

I use 2-step qPCR protocol: RNA extraction --> DNAse I treatment --> RT using MMLV-RT --> qPCR using cDNA as template.

I only quantify my starting RNA concentration, to employ the same amount (4 ug) for DNAse treatment in all samples. I do not perform any further quantification of RNA or cDNA.

Is this correct, or should I quantify RNA (post DNAse treatment) and/or synthesyzed cDNA, to assure equal concentration of all samples?

Thanks in advance.