mRNA isolation from monocytes
djoyce at receptor.pharm.uwa.edu.au
Thu Oct 10 01:40:27 EST 1996
In article <DyzBpw.Bxs at midway.uchicago.edu> kug1 at midway.uchicago.edu (jay kugler) writes:
>From: kug1 at midway.uchicago.edu (jay kugler)
>Subject: mRNA isolation from monocytes
>Message-ID: <DyzBpw.Bxs at midway.uchicago.edu>
>Sender: news at midway.uchicago.edu (News Administrator)
>Organization: The University of Chicago
>Date: Tue, 8 Oct 1996 22:56:20 GMT
>I'm trying to isolate mRNA from human peripheral monocytes, and am having
>no luck. I've been using Pharmacia's Quick-Prep micro kit. Can anyone
>who has experience isolating mRNA from phagocytic cells give me a hint
>on how to make this work successfully? I'm getting zero yield.
>Email is preferable, but any information is useful.
>Jay Kugler University of Chicago, Department of Pharm./Phys.
>kug1 at midway.uchicago.edu
We, too, have found that fresh human monocytes often yield degraded
RNA (total). After a day or two in culture, though, RNA extraction is
usually successful. During the first 24hr after purification, there is a
high rate of apoptosis, which may have something to do with it. The rate of
apoptosis is reported to be lessened by the presence of LPS or some
"inflammatory" cytokines. Perhaps the chances of getting good RNA are
improved if you are not scrupulous about preventing LPS contamination of
media, elutriator, etc. However, freedom from LPS contamination is important
for most work with monocytes.
I would be very interested if you find a way to successfully get good
quality mRNA from fresh human monocytes.
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