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