mRNA isolation from monocytes

[David Joyce]

In article <DyzBpw.Bxs at midway.uchicago.edu> kug1 at midway.uchicago.edu (jay kugler) writes:
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>From: kug1 at midway.uchicago.edu (jay kugler)
>Subject: mRNA isolation from monocytes
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>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.