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Ideas for determining poly(a) status of RNA - (Apr/04/2012 )

Hello, everyone.

I was wondering if I could get some input on how to best approach a particular problem. I'm a Master's student, and I'm helping supervise some undergraduate projects in the lab. We work on a (+)-sense RNA virus; particularly, I look at RNA secondary and tertiary structures and their implications during viral replication. Previous work in the field has demonstrated that the 3' end of the viral genome is blocked to polyadenylation and modification by T4 polynucleotide kinase. We have a working hypothesis as to why this is, and we have generated mutants toward this end.

I need help deciding which method would be most appropriate for determining whether the 3' end of the genome does become polyadenylated. After in vitro transcription, we could:
-Northern blot: use probes against the viral RNA core; expect to see smears in the lanes with poly(a) pol.
-Gel electrophoresis: would we be able to see the 3' modification on a denaturing formaldehyde-agarose gel?
-3' RACE: personally, I think this may be the best choice.

Is there any other way I can determine whether our mutants become polyadenylated? Thank you very much for your help!


And the answer (from lab meeting) was: urea-PAGE.


You could perform a PCR using a polydT primer and a primer that is specific for a region inside your virus genome. I would aim for a final product that is about 200 bp long (i.e. the forward primer binds 200 bp from the end of the virus genome), then just use a generic RT-PCR kit and run the product on a gel. This would not be quantitative but would tell you if you have any polyA there. You could also do it by QPCR but the probe would cost a bit. A separate set of QPCR primers and probe could also be run alongside the polyA set that binds to a known region within the virus genome. The ratio between this, and the one that binds the polyA, would give you a rough idea of the percentage that has a polyA compared to the total amount of virus genomes in the tube.