Storage of RNA

[Peter Gegenheimer]

In <37k1mp$jet at canopus.cc.umanitoba.ca>, hamel at cc.umanitoba.ca (Andre Hamel) writes:
>RNA should store well in sterile dH2O in freezer.
>
>We have MANY aliquots of total RNAs obtained from several types of
>specimens (feces, tissues, whole blood, etc, etc) ... ALL store WELL in
>-20*C PLUS used daily (frozen-thawed MANY times) ... used for RT-PCR ...
>ALL are still okay, right down to last bit used (most aliquots are 50 uL,
>with 1 uL used per reaction).
>
>If stored in formamide (such as used for northerns) ... simply ppt with
>alcohol and NaAc ... add NaAc to 0.3 M final conc and use 1-2 vol of
>isoprop or 2-3 vol of EtOH, mix well, incubate -20*C for at least 1 hr
>before harvesting pellet ... formamide will be washed away (remains in
>supernatant along with the alcohol).
>
>cheers,
>
>
>                            ********************
>Andre Hamel                                    email: hamel at cc.umanitoba.ca
>Manitoba Government                            tel: 204/945-7630
>Department of Agriculture                      FAX: 204/945-8062
>Veterinary Services Branch          
>Winnipeg, Manitoba, CANADA 
>                         ********************
>
>
>In article <sticknbd-121094224623 at 160.129.125.10> sticknbd at miranda.cc.vanderbilt.edu (B Stickney) writes:
>>In article <1994Oct7.192254.1 at molbiol.ox.ac.uk>, rhubner at molbiol.ox.ac.uk
>> > In article <ros.2.2E911FE4 at molbiol.uct.ac.za>, ros at molbiol.uct.ac.za
>
>--- stuff deleted ---
>> > > I have been storing RNA samples from both E.coli and Thiobacillus 
>>> > ferrooxidans in DEPC water or RNA storage buffer, 20mM sodium phosphate pH   
>>> > 6.5  1mM EDTA.  The T.ferrooxidans appears to be ok on a agarose gel, but no 
>>> > longer gives good results on a Northern Blot, and I presume this is due to 
>>> > degradation of the RNA.  I store the RNA at -70 C and thaw and refreeze it 
>>> > every time I use it, so this could also be causing problems.  I see some 
>>> > people store their RNA in formamide at -20 C, how do they then get rid of 
>>> > the formamide, when they want to do primer extension for example?   
>

There are two preferred ways to store RNA (take it from an old-timer).

1) Especially for smaller RNAs (< a few hundred nt), storage at -20 or -70 degrees in 
TE buffer or water works well.  There will be absolutely NO degradation IF the RNA 
itself is 100% pure (proteinase K-treated, etc, etc), all buffers and storage tubes 
are sterile (autoclaved), and you habitually use RNase-free technique (gloved hands, 
autoclaved or oven-baked reagents & glass+plasticware).  

2) The best way to store RNA is as an EtOH precipitate -- i.e., in 70% EtOH -- at -20 
or -70 deg.  To use, mix to resuspend, then remove the volume needed and spin down 
the ppt.  

DO remember that using gobs of RNases elsewhere in your lab -- such as for plasmid 
preps -- may create contamination.  If you want a REALLY RNase-free lab, handle 
RNases as you would radioisotopes:  work on a disposable benchliner & with disposable 
gloves; never weigh RNases (or proteases, etc.)  on a balance (to prevent traces of 
powder from flying loose), avoid any pipeting or vortexing manipulation that could 
release aerosols, and generally be aware of what you're doing.  

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|  Peter Gegenheimer            |  pgegen at kuhub.cc.ukans.edu             |
|  Departments of Biochemistry  |  voice: 913-864-3939                   |
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