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.