RNA isolation from whole blood
P.Rohde at mailbox.uq.edu.au
Sat Oct 4 05:57:54 EST 1997
Yes, I may be able to help.
Getting RNA with trizol from cell lines is a cinch. With biological
samples, when resuspending the RNA, USE an RNase inhibiter. I have
just used Promega RNasin, and it works. Put it on the RNA pellet first,
then add water or TE, *with* 10 mM (I guess) DTT so the enzyme works.
I use 80 U / 30 ul TE.
Also, with TRIreagent don't over do the reccomendations of sample:reagent
Despite the instructions also try doing *everything* on ice, (except
pelletdrying) and precip RNA for -20 C for 1 hr.
As far as detergents, TRIreagent doesn't go frothy - does this allow
for a guess?
If you want to try your faithful chom.&sacchi method (though I find TRi
reagent better for some reason (a secret ingrediant?), instead of using
solution D, add the GITC salt (1 ml blood => 0.5 g GITC, 7.2 ul/ml mercapto
EtOH, 1/10 vol 2M pH 4.0 acetate, equal vol acid/water phenol,
and chloroform followed by a few (bloody many) phenol chloroform extractns
If you can, do you pellet the blood (all cells) for TRI reagent extraction?
One more thing, In Australia, TRI reagent from Sigma is by FAR the
Please feed back, if you find anything.
jnakamot at ucla.edu (Jon Nakamoto) writes: >
> We're trying to analyze RNA expression patterns of human mixed leukocyte
> populations immediately after drawing the blood sample (i.e., we don't have
> time to let the samples sit, lyse the RBCs, separate out individual
> populations by adherence, etc.)
> Using reagents such as Trizol LS (with or without added acetic acid),
> TriReagent BD, yields from either whole blood (directly added to the
> reagent or from EDTA tubes) or from buffy coats (sucked out of EDTA tubes
> after a quick spin) have been uniformly terrible (RNA isolation from
> cultured lymphoblasts performed in parallel give good yields, so it's not a
> simple problem of lab klutziness).
> Any tips? I know there's a protocol using Catrimox, but the amount of
> Catrimox used relative to the volume of whole blood is huge.
> A long time ago I used to mix up my own guanidinium isothiocyanate-acid
> phenol solution (a la the Chomczynksi & Sacchi original recipe), drop the
> buffy coats into this, layer it all on a CsCl/EDTA cushion and spin it down
> in an ultracentrifuge -- got fair yields of good quality RNA at the bottom
> of the tube. I note that there was 10% sarcosyl in this GIT/acid phenol
> solution -- could this be making the difference (anyone know if Trizol and
> TriReagent include this stuff?)
> Thanks for your insights.
> Jon and Daina
> Jon Nakamoto, MD,PhD
> Assistant Professor, Pediatric Endocrinology
> Daina Dreimane, MD
> Fellow, Pediatric Endocrinology
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