RNA isolation from whole blood
paquetty at ERE.UMontreal.CA
Thu Oct 9 13:20:32 EST 1997
Rose-may.Delrue at fundp.ac.be (Rose-may Delrue) writes:
>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
>> Daina Dreimane, MD
>> Fellow, Pediatric Endocrinology
>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,
>Pascal.Lestrate at fundp.ac.be
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.
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