RNA isolation from whole blood

Jon Nakamoto jnakamot at ucla.edu
Thu Oct 2 11:46:37 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



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