RNA storing/RT PCR using oligo dT - (Feb/09/2006 )
Hi everyone, i've done a RNA extraction three weeks ago, stored in DEPC treated water at -80°C... how long will it last? what are the better choices to store RNA for long time and either being suitable for RT PCR?
Now the real problem: i need to amplify a gene with unknown 3', i tried using the oligo dT supplied in the kit (SuperScript III Cells Direct cDNA synthesis, Invitrogen) as a reverse primer and a GSP as forward (Tm 56.9), i have done annealing at 50° 30'', amplification using Pfx at 68° 3', no band appeared. Any idea about negative results? perhaps my RNA is already totally degraded (despite i saw a huge smear in one of the samples) or oligo dT unbinds at 68°? do i have to use 3' RACE necessarily?
Thank you very much,
For RNA storage, you can go to the Ambion, Applied Biosystems, and Qiagen websites. They have some good technical bulletins there. Here is the Ambion website:
Sorry, but I can't really help with the rest of your question. Have you tried testing your RNA by itself for degradation?\
You would need to generate cDNA using oligo dT as the primer
Then for PCR use your GSP and a poly A primer. You can't use oligo dT as a primer in PCR of cDNA as the cDNA has a poly T "tail", rather than a poly A tail
I would recommend converting your RNA into cDNA as soon as possible and storing it that way. cDNA is much more stable and then you can worry lesss
Well guys, thanks for helping. I forgot to say that i already made the cDNA from RNA, RNA 3 weeks old in DEPC-treated MQ water but even so... now, do you think that doing PCR with oligo AAA (this would be coding sequence, hence, anneals in cDNA), i could get some stop codon? anyways, thank you again, will see what can i do.
For 3’RACE, it is better using polyT anchor primer, for instance wi:
..XXXXXXXXXXXT(n)VN, which V is A/G/C, N is A/T/G/C and ..XXXXX.. is seqeuences used for reverse primer along with your Gene specific forward primer.
PolyT can be used for cDNA synthesis and might not work in PCR amplification.
Some times even degraded RNA still can be used for 3’RACE although efficiency is lower than intact RNA…