Re: detection of mRNA by flow cytometry

SPERFETTO (sperfetto@hiv.hjf.org)
Thu, 16 Nov 1995 08:49:19 -0500

mRNA will be most difficult because in most cases it is rapidly degraded.
However, most of our probe work has been with PCR amplified HIV-gag DNA. Two
approaches were attempted a) the direct probe with FITC and b) the direct
incorporation of UTP-digoxigenin within the PCR products. We were not able to
get the direct probe sensitivity high enough over background but Kent Lohman
from BDIS has been more successful. However, we were successful with DIG
incorporation using a secondary labeling with anti-DIG-FITC. To actually bring
the sensitivity up to a reasonable level we quenched much of the autofluorecence
by using 0.05% of evans blue. This dye will stain the cytoplasm red and cells
can be detected using a standard red filter setup, other than that the chemistry
is unknown. With this technique we were able to detect 1:1000 but it is still
under development.

Stephen P. Perfetto
Department of HIV Disease Prevention
Walter Reed Army Institute of Research
1600 East Gide Drive
Rockville, MD. 20850

_______________________________________________________________________________
Subject: detection of mRNA by flow cytometry
From: h-petrie@ski.mskcc.org (Dr. Howard Petrie) at Internet_Gateway
Date: 11/15/95 11:42 AM

Dear fellow cytometrists: I am interested in attempting to detect mRNA on
a single cell basis by in situ staining and flow cytometry. I am aware of
a few papers in the literature (globin message), but they are a little
dates now. I am wondering if anyone could share their experience regarding
similar applications. Are biotinylated riboprobes most likely to be
useful? Which biotinylated nucleotides have been used? How can signal
intensity/ background ratios be maximized? I will repost a summary in the
case of multiple responses. Thanks in advance. HTP

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