Reverse-transcription of UV-damaged RNA - (Jan/28/2010 )
Hi all,
We have to UV-crosslink RNA in our procedure a few steps prior to cloning, and have found it to be difficult to get much cDNA. Does anyone have any ideas on how to convert UV-damaged RNA into cDNA?
Is there a way to repair/prevent pyrimidine dimers etc. generated by UV? Or is there a robust RT that can jump over damage? I have heard that HIV RT can proceed through RNA lesions (with low fidelity) - are there any commercially available RTs that can handle them? Fidelity (while desirable) is not our prime concern.
maybe you can try to use random hexamers, as primers, for cDNA synthesis...
microphobe on Jan 28 2010, 01:35 AM said:
We have to UV-crosslink RNA in our procedure a few steps prior to cloning, and have found it to be difficult to get much cDNA. Does anyone have any ideas on how to convert UV-damaged RNA into cDNA?
Is there a way to repair/prevent pyrimidine dimers etc. generated by UV? Or is there a robust RT that can jump over damage? I have heard that HIV RT can proceed through RNA lesions (with low fidelity) - are there any commercially available RTs that can handle them? Fidelity (while desirable) is not our prime concern.
maybe you can try to use random hexamers, as primers, for cDNA synthesis...
Depends on the RNA sample - for pure mRNA it may be a way to go for, but when you have total RNA, the random hexamer primers would attach to all RNA molecules, including all the rRNA stuff
depends what you want to do with the RNA. If you want to do qPCR, microarray, ....? if so, this would not be a problem