RNA stability and storage
Dr. Peter Gegenheimer
pgegen at ku.nolospamare.edu
Sat Jul 7 17:32:02 EST 2001
On Fri, 6 Jul 2001 16:33:03, campanellj at mail.montclair.edu ("Dr. James
J. Campanella") wrote:
| I'm just wondering how most people store their RNA extracts.
| Obviously freeze-thawing is not a worry in long-term storage,
| but if you are using your RNA extracts every day and
| storing them frozen, how do you ensure that you do not
| lose much RNA to degradation? Sorry if the question is a bit
| naive, but I have mostly dealt with DNA in the past . . .
In principle, if the RNA is stored in sterile buffer at pH <7 (i.e.,
NaOAc, ph 5.5) and with 0.1 - 1.0 mM EDTA, it will be stable
"indefinitely." Another procedure has been commercialized by Ambion:
store the RNA in 1 mM Na-citrate buffer, pH 6.4. Citrate (like EDTA) is
a chelator of divalent cations which generate hyroxide ions that attack
phosphodiester bonds. A low pH also reduces the concentration of free
hydroxide, and RNA is stable between pH 5 and 6.
In practice, people who are very concerned about the quality of, say, an
mRNA prep to be used as a source for cDNA or mapping, will store the RNA
as an ethanol precipitate. I don't know whether this is any better than
citrate or EDTA, but it used to be regarded as the "gold standard" for
Measure the concentration of RNA (by any means you like). Ppt the RNA
from 0.2 M NaOAc (pH 5.5) and 2.0 - 2.5 vol absolute EtOH (or anhydrous
EtOH: 95% EtOH, 5% isopropanol). Do the precipitation at 4C or -20C (no
colder). The ppt can be stored at -20 or -70. Record the final
concentration of RNA in the ppt. When you want to use some of the RNA,
mix the EtOH solution to restore the RNA to a uniform suspension.
Withdraw an aliquot of the required volume and centrifuge it (say 10
min, 10K x g). Rinse the pellet and proceed as usual.
| Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: 785-864-5321 |
| Department of | PGegen at KU.nospam.edu |
| Molecular Biosciences | http://rnaworld.bio.ukans.edu/ |
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