How much proportion of the total RNA extracted by Trizol is nuclear RNA ?

If the total amount of the RNA extracted by Trizol is 3μg, and the transcript (the primary transcript of a highly expressed miRNA) of interest is generally considered to be in the nulceus, then is it possible to detect it with qPCR and PCR? To a more general question: how much proportion of the total RNA extracted by Trizol could be nuclear RNA ?

Biochemistry

RNA Extraction

Real-Time PCR

Conventional PCR

Jack M Gallup

Vaxxinova/Newport Laboratories, Worthington, MN

Adding to Jörg Krummheuer above, also be absolutely certain you have a highly effective DNase treatment in place so that marginal gDNA contamination doesn't get confused with positve qPCR signal for pre-mRNA species when using the exon-intron border-spanning primers; the same complemetary sequence(s) would exist in the gDNA. Adding just RNA to a qPCR or PCR would let you know if gDNA contamination is still there... (as a control)... and, if no signal there, then whatever signal you get otherwise in your cDNA-based qPCR/RT-qPCR or PCReactions can only be ascribed to the presence of pre-mRNA-derived cDNA. With the primers in place that Jörg has mentioned, this could be your best bet. And back to the original point: I believe Trizol is thought to be chaotropic enough (coupled with ample homogenization) to dismantle the nuclear membrane and release the goods; cytosolic tRNA is included as well. Some estimates have it that 83% of Total RNA is rRNA wherein: 20% of Total RNA is 18S rRNA, 63% of Total RNA is 28S, 5.8S and 5S rRNA, and ~2 to 2.5% is mRNA. This leaves about 14.5-15% to be tRNA and nuclear pre-mRNA; which makes Jörg's 5-10% figure (for fraction of Total RNA that is 'nuclear RNA') very sound, in eucaryotes.

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