mRNA storage

Peter Gegenheimer peterg at rnaworld.bio.ukans.edu
Sat Aug 27 14:56:46 EST 1994


In <33fusb$31q at mserv1.dl.ac.uk>, "J.P. Hays" <jph5 at leicester.ac.uk> writes:
>Dear Colleagues,
>
>  I am having great difficulty in maintaining stocks of "viable", 
>(i.e RT-PCRable), viral mRNA, and would be very grateful of any suggestions.
>
>Initially, I extract viral positive sense RNA using RNAzol, (Biogenesis), and
>store the product in 50 microlitres TE buffer with 0.25 microlitres RNasin,
>(Promega). The components for the TE buffer are individually produced as stock
>solutions, autoclaved, and mixed when required. The RNA is stored in DEPC
>treated, sterilised, Eppendorfs, (though the glassware in which the TE buffer
>is stored and the water used in the TE buffer components are not DEPC treated).
>RT-PCR on freshly isolated RNA works well, but after 2 days at -20oC
>the RNA seems to have disappeared! 
>

First, the RNA must be RIGOROUSLY deproteinized by phenol extraction or, preferably, proteinase
K digestion. I know of no other methods that will remove all traces of RNase from the 
biological sample itself!
Second, DO oven-bake all glassware and autoclave all solutions.
Third, make sure that ALL the DEPC is gone before adding RNA solutions -- concentrated DEPC 
will quickly degrade RNA.

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