In Article <lK%m3.13966$8v6.678204 at carnaval.risq.qc.ca>, "Paula Lavery"
<plaver at po-box.mcgill.ca> wrote:
>We are trying to isolate RNA from patients' blood samples. So far, I have
>been lysing RBC from buffy coats with ammonium chloride solution and storing
>the WBC in DEPC-PBS at -70C. (RNA isolation is done later in another lab.)
>The RNA yield is very low from these samples. Is anyone else isolating RNA
>from blood? I'd appreciate any help or hints with this. Thanks!
I see one potential problem with what you are doing. If DEPC-PBS is PBS
that has been treated with DEPC and then autoclaved, that will not have any
DEPC remaining, and freezing and thawing the cells will allow the cellular
RNases to chew up your RNA. If you have PBS with fresh DEPC, it is toxic,
will kill the cells without quickly penetrating in sufficient quantities to
inhibi the endogenous RNases; same result.
I usually get tissues as fresh as possible and get them homogenized into
a strongly reducing and denaturing buffer to stop all RNases as fast as
possible. I would think that might be a more stable situation than what you
describe. Of course, the best bet is to purify the RNA immediately, if that
is possible.
Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton, Alberta T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca
