Base Composition Determination for RNA - Not sequence determination. Molar ratios: AGCU & X (May/12/2008 )
Does anyone have a protocol for determining base composition for RNA? I want a method to degrade RNA to look at nucleotide content, so I can see if certain nucleotide analogs are getting incorporated into RNA. Such methods used to be common before reverse transcription and DNA sequencing were invented. I have found several references from the 1960s, but I'd like a modern protocol that requires less input RNA and analysis by HPLC, if possible.
Any old timers out there with a good protocol?
How about trying a partial digestion of the RNA and separation based on MW through HPLC. You would need to run a reference RNA through to give you an indication of the normal MW.
Sorry, that is as good as I can come up with.
That might be a possibility, if the nucleotide analogs differ in mw significantly from the natural nucs. and the incorporation is significantly high. But I think I should start out with total digestion of the RNA to look for the analog. I have found some TLC methods, using PEI plates, that require labeling cells with 32P. It would be nice to do a cold experiment.