Which temperature in Reverse Transcription? - (Jul/12/2005 )
How do we know at which temperature we should use for cDNA synthesis when we use random, specific and Oligo(d)T primers in RT? What decides this temperature?
I'm new to bioforum. I have used QIAGEN's sensiscript RT. I got good cDNA with oligo dT when I used RT at 37deg for 2hrs. I think all RTs work at 37deg,if I'm not wrong.
i used 37 degree for RT from NEB, but 42 degree for RT from invitrogen. you can use 42 degree for RT from NEB as well. You should read the protocol provided by the company before you carry out rt-PCR
I think the temperature depends on the reverse transcriptase mainly. I use AMV reverse transcriptase (which is part of the kit Improm II from promega) at 42. In spite at lower temperatures there would be less denatured AMV, I prefer a higher temp to avoid RNA secundary structure.
It depends on the kit (enzyme) you are using. I think most work @ 37-42°C, but for instance invitrogens superscript III is fully active @ 55, Roche's transcriptor @ 50, invitrogen's thermo-X or thermo-script even @ 70°C, which prevents to some extent mispriming and also secondary RNA structure (I got good results with superscript and transcriptor myself). I think it's best to read the manufacturer's instructions or maybe do a little optimisation yourself...