Dear all,
does anyone have experience how the cycling conditions influence cDNA synthesis using the Taqman Reverse Transcription Reagents with random hexamers for qRT-PCR applications. For years I successfully use the following cycling conditions: 10 min 25°C, 1h 48°C, 5 min 95°C. When I checked the current protocol from the supplier they recommend 30 min 37°C as the middle step.
Now I wonder, if the yield could be even higher if I use the recommended 37°C instead of 48°C or if maybe one of the temperature favors a bias of different transcripts. Does anyone have experience and could comment on this?
Thanks a lot!
Gertrud
Dear Gertrud,
You have to take into account that
1) melting temperature of each primer at fixed conditions depends primarily on its concentration, length and GC-content,
2) random hexamers are the complex mixture of very short DNA primers with GC-content ranging from 0% to 100%,
3) even the hexamers with 100% GC have relatively low melting temperatures (even at highest concentrations typically used for RT),
and 4) although the overall concentration of random hexamers may look high, the concentration of each individual variant of a hexamer in the mixture is obviously much lower.
Taken together, that means that the higher is the temperature of your RT, the smaller is the fraction of hexamers that really work as primers at that temperature. If you target is very AT-rich, the yield of cDNA may dramatically fall if you use random hexamers at high temperatures. On the other hand, increase of the RT temperature may help to overcome the problems with secondary structures in your RNA, such as hairpins. The two-step RT protocols (like your 25C and then 48C) are usually intended to deal with hairpin problem: annealing and beginning of elongation starts at 25C, then the duplex of cDNA/RNA becomes more stable and further cDNA elongation may proceed at higher temperatures that favor the melting of secondary RNA structures. For different purposes I have used different two-step RT protocols with temperatures at the first step ranging from 10C to 25C and at the second step from 35C to 65C. In my hands it resulted in smaller bias of different transcripts compared to one-step protocols.
Hope it will help)
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