Reverse transcribing organically contaminated RNA - (May/18/2009 )
For one of my RNA edxtractions using Qiagens RNEasy minikit, I got low abundancy, contaminated RNA. My 260/280 looks good at 2.01 but my 260/230 is .53. I don't know why because I follow the protocol and do it the same everytime and all my other RNA extractions look good. I even rocked the RPE buffer around in the tube because I read that sometimes the RLT lysis buffer can stick to the top of the top then come off during elutions. Anyway, I was considering cleaning it up again using the kit but my concentration is 41.2 ng/ul. I would lose a lot cleaning it up again. So my question is would it be alright to reverse transcribe this RNA or would the organics in it destroy the reverse transciptase enzyme?
Sounds like you might have some residual ethanol in your RNA eluate. If you have access to a spin-vac concentrator, you can evaporate the ethanol and concentrate your sample prior to RT, and you should be fine.
In the future, you can make a couple of simple modifications of the recommended protocol to eliminate the EtOH contamination, that will also increase yield and purity. Prior to eluting the RNA at in the final step, make sure to let the column stand at room temperature for 5-10 minutes with the cap open to allow the ethanol from the RPE to evaporate. This should help reduce your A230, and may also increase your yield, as the residual ethanol casues some RNA to remain bound to the column media. You can also try warming the eluent ( RNase-free TE or water) to 50-60`C and extend the incubation time out to 2-3 minutes which also helps increase yield, without increasing degradation. Hope that helps.
Not a bad idea, thanks.