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Total RNA, mRNA quantitation by qPCR?
I'm assuming there's a fairly fundamental reason why this isn't done, at least I haven't been able to find any informative reference so far...
I want to accurately quantify DNA, total RNA (~rRNA) and mRNA across many, very small samples (mutants and timecourse). DNA, easy enough, but for RNA I don't see why I can't just RT using either random or oligo(dT) primers, and then run the qPCR rxn with random primers (I have random 15mers, Tm very low at 40C though). I don't want to make ANY assumption about 'reference' mRNA here - the conditions are potentially highly dynamic. Possibly I could go for specific primers to the major rRNA species, but that would be a bit of a pain.
Anyone else tried this or know of a reason it would not work?
I've done a single trial run, 10ul rxns with 10-fold dilutions from 10ng down to 10pg, but no take-off whatsoever, nothing on the melt. I might try larger rxns, maybe 20mer primers, higher [primer]. It would be very nice if this would work, but perhaps I'm being too simplistic.
|dna , mrna , qpcr , quantitation , rna , rrna , total|
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