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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-

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

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,


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 65C 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 (:

-lna-