RNA extraction after sorting for intracellular antigen

Guy Hermans Guy.Hermans at ablynx.com
Fri Nov 25 05:46:06 EST 2005


Hi Max,

There's a few references to the use of RNAlater (Ambion) for cell fixation
and RNA preservation prior to cell sorting, with recovery of functional RNA
after sort. Check out biotechniques 2002 32 888.

Guy

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-----Original Message-----
From: Max Warncke [mailto:max.warncke at uniklinik-freiburg.de] 
Sent: Thursday, November 24, 2005 10:07 AM
To: cyto-inbox
Subject: RNA extraction after sorting for intracellular antigen


Hi,

we are looking for a method to extract RNA from lymphocytes which were fixed
and permeabilized and stained for an intracellular antigen. We have some
problems in doing so with normal Formaldehyde/Saponin protocol. We want to
use the RNA for spectratyping. If we try to extract RNA from fixed specimen
we do not obtain any RNA (not even degraded one) and it looks like the whole
RNA is just leaking out of the cell. Do we have to treat our buffers with
DEPC or ß-ME? Are they better methods for fixing the cells or crosslinkin
the RNA to the cell. Any suggestions would be helpful.

Thanks

Max



	____________________________________

	Max Warncke, Dipl. Biol.

	University Freiburg Medical Center
	Medicin I
	AG Veelken

	Lab. Nothnagel / 5.OG
	Breisacherstr. 117
	79106 Freiburg

	fon: +49.761.270- 7196
	fax: +49.761.270- 7177

	____________________________________


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