Hi everyone,

After reverse transcription, I need to measure cDNA:RNA hybrid quantification befor qPCR analysis. I do not know, if I should measure ssDNA or dsDNA? Many people here had similar question and the advice was not to measure this hybrid molecule, just measure RNA concentration and use the same amount for RT reaction and afterthat perform qPCR with reference gene/genes. However, when I want to test reference gene, I have to measure cDNA concentration and use the same concentration for every sample in qPCR. RT reaction is not 100% for all samples, also after RT, cDNA purification is important (Susler and Steindler 2005, and also my experience - bad amplification efficiency, when not purified) and after purification (column-based purification, Zymoclean) each sample has different concentration. I used ssDNA assay kit (qubit), but I am not sure. Thanks a lot.