RNA stability

susanne srohrerNOsrSPAM at immv.unizh.ch.invalid
Fri Nov 19 10:29:47 EST 1999


Hi

I have been isolating total RNA from bacteria and storing it in dH2O at
-80. Now I read on www.nwfsc.noaa.gov/protocols/ that it is a good idea
to dissolve the RNA in formamide. It is said to keep it fully
denatured, can be stored at 4°C overnight or indefinitely at -20. A
first try Northern Blot looks different, possibly better than the
earlier versions, I would even assume the RNA is "more denatured" than
before, see below.
Anyone tried this before? Does Formamide have to be of molecular
biology grade, eg. deionized? what adverse effects would "bad"
Formamide have?
Problem 2:
Does anyone have experience with signals around the rRNA bands? could
it be from interactions between mRNAs and rRNA, if it is not fully
denatured? or could degradation products interact with rRNA?
I have been detecting a major band just below the 16S rRNA band, and a
minor fraction at the expected size (5kb). With the above formamide
method, a strong 5kb band was detected, and the lower band was gone.

Thanks for suggestions

Susanne Rohrer, Uni Zurich



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