heating rna before rt reaction? - (Oct/22/2009 )

hi, i have a question about RT reaction:
i am using invitrogen vilo cdna synthesis kit for rt reaction. on the instruction it just says add rna sample, does not mention heating.

however i read on some web page saying that rna sample should be heated briefly before rt reaction.

i am just wondering if this is necessary? if yes, what is the purpose for it and how long/hot do you normally heat it?

thanks,

supermr@tamu on Oct 23 2009, 01:12 AM said:

Hi,
if I'm not mistaken, the heating is needed to "melt" all the secondary structures and to anneal the primers (Hexa or oligoT) or at least to give them a better chance to interact with RNA. In the standard Invitrogen kit they usually recommended to heat the mix of RNA, primers and dNTPs at 65°C for 5 min before the reaction. However, in general the reaction can work well without heating as well if your target RNA does not have strong self-complementary sites. Before using Invitrogen I was using the home-made reverse transcriptase and never did the heating step, but all was working quite well. The point is that some enzymes can loose their activity after heating, so I think that if you decide to heat - do it before the revertase addition. Anyway, if the protocol says that there is no need for heating, than it seems like you can abandom it (: