mRNA storage

[Lisa J Brunet]

J.P. Hays (jph5 at leicester.ac.uk) wrote:
: 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! 

: Thanks for any advice!


: John Hays

: jph5 at le.ac.uk
RNasin requires DTT to remain active. Otherwise it degrades releasing 
RNase. You may try adding DTT to your RNA stocks or leaving out the 
RNasin altogether. I just store my RNA in TE at -80 with no problems.