RNA isolation from whole blood

[Jon Nakamoto]

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
UCLA

Daina Dreimane, MD
Fellow, Pediatric Endocrinology
UCLA

[Dom Spinella]

Well, I know you don't want to hear this, but I would at least lyse the
red cells first -- they have lots of crud that appears to interfere with
subsequent isolation and manipulation of nucleic acid. I bet if you just
resuspend your buffy coat in a few ml of Tris-buffered ammonium chloride
for a few minutes, (e-mail me if you need a recipe) then wash the
leukocyte pellet a few times in a microfuge (at low speed) with PBS or
TBS, any procedure you use for RNA isolation will work much better. It
really doesn't take much time and its well worth the effort. Just a
thought. -- Dom Spinella

[Paul Rohde]

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
cheapest.

Please feed back, if you find anything.

[Rose-may Delrue]

In article (Dans l'article)
<jnakamot-ya023680...@news.ucla.edu>, jnak...@ucla.edu
(Jon Nakamoto) wrote (écrivait) :

The blood is full of RNAse and it's really hard to extract a hight quality
mRNA from blood sample, We did it next year by using Ultraspec (Biotecx, USA)
and we got nice result,
Good luck,
Pascal
Pascal....@fundp.ac.be

[Paquette Yves]

Rose-ma...@fundp.ac.be (Rose-may Delrue) writes:

If you can analyze your RNA with RNase protection, you can do it on whole blood using the Ambion Direct Protect kit. You just mix your blood sample with an equal volume of lysis buffer, vortex and add your RNA probe. After hybridization, when you add the Rnase digestion buffer, you do get a red hemoglobin precipitate, but it is digested away at the next step with proteinase K.
I tried that technique to measure IL8 mRNA induction in whole human blood and got nice clean results.
Good luck !

Yves Paqquette Ph.D.
Hopital Maisonneuve-Rosemont
Montreal, Quebec