I'm very embarrassed to admit this, but I don't understand how random hexamer primers (RHP) work in reverse transcription. I made RT with gene-specific or oligo-dT oligos hundreds of times, the whole idea is absolutely clear. But when we come to RHP...

Okay, let's say we have set of random hexamers, the most downstream one (green on the upper picture) anneals to our RNA template and serves as a primer for reverse transcriptase. But what about others, annealing somewhere upstream (purple on the picture)? What happens then RT enzyme reaches them, why don’t they (especially GC-rich ones) interfere with revertase movement? At least in case of PCR such oligos annealing inside the amplified region effectively block the amplification.

On the other hand, if all of these random hexamers are capable of priming reverse transcription, in the end we will have a whole bunch of short cDNA fragments, barely usable for subsequent PCR amplification.

I’m afraid I miss something very important (and simple). I would greatly appreciate any clarification!

Stan