How to identify an unknown RNA? - (Aug/11/2006 )

Hello. When I incubate an uncharacterized enzyme, probably a poly(A) polymerase, with alpha 32P- ATP I find that specific RNAs are labeled when they are run out on polyacrylamide gels (100-300bp). I've tried biotin-labeled ATP analogs but they are inefficiently incorporated into RNA with this enzyme and because of the sharpness of the bands, it would seem that only one up to a few hot adenosines are being added to the substrate. Does anyone have any other ideas that would allow me to isolate these specific RNAs? Thanks.

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What about an RT step? You could make first strand cDNA and then try to hybridise to a small array.

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Thanks for the reply. The problem with the RT step is that I cannot selectively RT the hot band. I assume that there are plenty of other RNAs that would also be made into cDNA. I like the idea of using the hot labeled RNA as a probe on an array but I would have to use an Affy genome array to make sure I got full coverage of all RNAs (mi, tRNA, rRNA, etc). And I don't know if a fluorescent label would be as sensitive as 32P.

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