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Date: Wed, 14 Mar 2007 15:06:40 -0400 (EDT)
Subject: Re: intracelular staining and RNA extraction
From: rrabin@helix.nih.gov
To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
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Don't know if this helps, but. . .

It is important to note that paraformaldehyde X-linking, which cross-links
methyl groups, can be reversed.  (Gluteraldehyde x-links free amines and
cannot be reversed).  Whether that means that the mRNA can be captured, I
don't know.  Another issue is whether the RNA is lost with the
permeabilization.

ron

> Greatings to everyone,
>
> I’m sorting B-lymphocyte populations in a FACS Vantage using four colors
> staining. I’ve got to permeabilize cells for intracellular staining and
> I’m testing cell fixation/permeabilization reagents. The reason is I
> have to extract RNA from those cells with array quality. The usual
> fixation/permeabilization reagents contained paraformaldehide and their
> cross-linking propriety didn’t allow RNA extraction. I’ve already
> tried to decrease paraformaldehide concentration but the result was the
> same.
>
> I also test organic fixation/permeabilization reagents like ethanol but my
> flow images were really bad.
>
> I found some papers where they refer Permeacyte (Bio-E) as a non-cross
> linking reagent that will probably suits. I try to find out who sells this
> product but seems that it is out of the market. Then another product named
> Permeafix (Orthon Diagnosis) will probably act the same way. The latter
> one no it’s no longer commercialized. Looks like Permiflow it’s the
> new
> one that substitutes those reagents.
>
> I test Permiflow and it goes perfect for my flow analysis but I still
> can’t extract RNA.
>
> Does anybody know of some reagent, staining procedure that fulfills my
> objective?
>
> Thank you very much for your help,
> Maria Joao Baptista
>
>