3 replies to this topic

[microphobe]

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.

[susanna]

maybe you can try to use random hexamers, as primers, for cDNA synthesis...

microphobe, on Jan 28 2010, 01:35 AM, said:

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.

[array75]

[quote name='susanna' date='Feb 10 2010, 05:08 PM' post='58375']
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

[susanna]

depends what you want to do with the RNA. If you want to do qPCR, microarray, ....? if so, this would not be a problem