RNA storage
Dennis
Does anyone have experience storing RNA at 4 degrees for long periods of
time? We normally store RNA at -80 long term, but I have recently heard
that storing RNA at 4 degrees may improve reproducibility in
quantification of RNA by OD260.
thanks for your time
DL
chr...@dfrc.wisc.edu
We mistakinly stored RNA at -20 C once for a few days and lost most of
it. Have never heard of 4 C storage, but you can test it on YOUR
samples if you want. Let me/us know!
chris
Eric Lader
We do long term stability tests of our total RNA at 37 degrees for two
days with no visible degradation. Total RNA can be stored forever at -20
degrees in TE.
Kevin Mulcahy
We have never attempted to store RNA at 4šC for long periods. However,
as regards storing at temperatures less than 4šC, we always
re-precipitate our stock RNAs after use (using ethanol and salt) and
store at -70šC. We have heard that this prevents possible freeze-thaw
degradation of RNA. Such RNA degradation may occur with dissolved RNA
when freeze-thawed a number of times.
Kev.
Mathew Traini
Kevin Mulcahy <K.Mu...@sheffield.ac.uk> wrote:
I don't know if this is such a problem for small RNAs, such as
ribozymes. We routinely store ribozymes (30-40mers) at -20C,
in autoclaved milli-q water, (labelled with p32), and notice
very little (if any) problems with degradation. These RNAs
were produced via solid phase synthesis, so obviously there is
no chance of contamination by anything cellular, which may be
a problem with say, mRNA extracted from a cell.
Mat.
Mathew Traini
Just another thought....
Was the storage buffer at a neutral pH?? At a basic pH,
cleavage at the 2' OH group will occur, giving you a nice
looking ladder of fragments. I don't know how much OH-
catalysed cleavage would occur at such a low temperature, ie: 4
degrees C though.
Mat.
Hiranya Roychowdhury
At 08:40 AM 11/5/96 GMT, Kevin Mulcahy wrote:
>We have never attempted to store RNA at 4=9AC for long periods. However,=20
>as regards storing at temperatures less than 4=9AC, we always=20
>re-precipitate our stock RNAs after use (using ethanol and salt) and=20
>store at -70=9AC. We have heard that this prevents possible freeze-thaw=20
>degradation of RNA. Such RNA degradation may occur with dissolved RNA=20
>when freeze-thawed a number of times.
>
>Kev.
>
>
>
>
The way I store my RNA sample is as follows:
I estimate the concentration of the RNA spectrophotometrically and then
store the standard RNA solution in 66.66% ethanol (ie. 2 vols) at -70 C. For
each use, I aliquote the required amount and add either NaCl or NaOAc to ppt
the RNA for 15 min at -70 C.=20
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroy...@nmsu.edu
Juan Pablo Martinez-Soriano
At 13:21 11/5/96, Dr. Hiranya Sankar Roychowdhury wrote:
> The way I store my RNA sample is as follows:
>
> I estimate the concentration of the RNA spectrophotometrically and then
> store the standard RNA solution in 66.66% ethanol (ie. 2 vols) at -70 C.
66.66% ethanol, then the rest is water?
If this is the case, RNA will be in solution and exposed to any contaminant
RNAse you would have in the water. The rationale of using salts is
precisely that.. to take the RNA (or DNA) out of solution and making it
"inert" to any nuclease action. Am I correct or did mess it badly?
> For
> each use, I aliquote the required amount and add either NaCl or NaOAc to ppt
> the RNA for 15 min at -70 C.=20
We take OD readings to estimate concentration, then precipitate with salted
ethanol (95%) and store at -20 or -70 C. When needed, we mix well the tube,
take an aliquote of an estimated amount, and proceed to spin and resuspend.
Best regards from Mexico
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Hiranya Roychowdhury
At 06:16 PM 11/5/96 -0600, Juan Pablo Martinez-Soriano wrote:
>At 13:21 11/5/96, Dr. Hiranya Sankar Roychowdhury wrote:
>
>> The way I store my RNA sample is as follows:
>>
>> I estimate the concentration of the RNA spectrophotometrically and then
>> store the standard RNA solution in 66.66% ethanol (ie. 2 vols) at -70 C.
>
>66.66% ethanol, then the rest is water?
Good Guess!
>If this is the case, RNA will be in solution and exposed to any contaminant
>RNAse you would have in the water.
If RNase is present, it will destroy the RNA even in its ppt. form over
time. While preparing and storing RNA, we assume that everything coming in
contact with the sample is free of RNase.
>The rationale of using salts is
>precisely that.. to take the RNA (or DNA) out of solution and making it
>"inert" to any nuclease action. Am I correct or did mess it badly?
You are partially correct. But, while DNase activity may be mostly arrested
while frozen, RNase activity has been known to go on (albeit at a vv slow
rate) even at -20 C. The time it takes to thaw a sample tube of RNA from
-70 to 0 C is sufficient to allow significant RNA loss if there is RNase
already present in the sample. Theoretically, one should be able to store an
RNase-free sample of RNA in buffered solution at room temperature.
>> For
>> each use, I aliquote the required amount and add either NaCl or NaOAc to ppt
>> the RNA for 15 min at -70 C.=20
>
>We take OD readings to estimate concentration, then precipitate with salted
>ethanol (95%) and store at -20 or -70 C. When needed, we mix well the tube,
>take an aliquote of an estimated amount, and proceed to spin and resuspend.
Each to his own device.
Hiranya