I have a reverse transcription 101 question! Does anyone know what happens to the cDNA/RNA hybrid molecules during a reverse transcription reaction? What I'm asking is, after a reverse transcriptase (RTase) lands on a mRNA molecule and synthesize the first-strand cDNA molecule, does the cDNA/RNA hybrid molecule stay as a hybrid until the end (i.e. it's impossible for that mRNA molecule be used as a template again), or does the cDNA/RNA hybrid molecule simply fall apart and the mRNA is available as a template to make more cDNA molecules?
The reason I ask this is because I'm trying to develop a digital PCR assay that would allow me to quantitate the amount of mRNA in my samples in an absolute quantification manner. I got the digital PCR part pretty well validated already. However, unless there's a cDNA synthesis system that would trully copy 1 RNA molecule into 1 cDNA molecule (and not more), then there's really no way to quantitate the absolute copy number of mRNA molecules...
Thanks for your help! I appreciate any thoughts and advice on this!