I have in vitro transcribed a collection of RNAs of 60-100 nt with a substantial proportion of double strandedness (similar to pre-miRNAs). After column-based purification to remove free nucleotides I read the concentrations using a spectrophotometric OD260 method (nanodrop) and got a set of readings in the middle of the linear range. To double check that there was no degradation, I ran out equal amounts of RNA on an agarose gel impregnated with SYBR Green, only to discover very different intensities of bands. Suspecting free nucleotide carry-over I turned to a fluorescent-based method for corroboration (Qubit) as the dye only binds RNA and not NTPs. I got readings that were in total discord with both the nanodrop and the gel. I suspect that since SYBR dyes and Qubit fluorophor are intercalating, they will be very affected by the degree of secondary structure. But then again, OD260 is affected by it too. Are there any methods to measure RNA concentration that are unaffected by structure?
Andrei, thanks for the response. The RNA is in-vitro synthesised, so I know there is substantial but imperfect self-complementarity (stem-loop with mismatches and bulges). The peak that I observe on UV spectrophotometry is single, so I suspect the RNA is completely folded.
Acridine orange is an interesting idea - I will read more.
Thanks again!
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