RNA isolation from whole blood

Dom Spinella dspinella at chugaibio.com
Thu Oct 2 14:49:40 EST 1997


> 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


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



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