RNA isolation from whole blood

Rose-may Delrue Rose-may.Delrue at fundp.ac.be
Mon Oct 6 07:19:02 EST 1997


In article (Dans l'article)
<jnakamot-ya023680000210970946370001 at news.ucla.edu>, jnakamot at ucla.edu
(Jon Nakamoto) wrote (écrivait) :

> 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
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.Lestrate at fundp.ac.be



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