measurement of mRNA-stability?

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Mar 3 12:59:48 EST 2000


>Hi everyone,
>does anyone know how to measure the stability or halflife of mRNA? How
>can I measure the length of the poly(A)-tail? Any tips are welcome.
>
>With best regards.
>
>Nils
>
>

The best ways to measure mRNA stability depends somewhat on what system you
are using, i.e. plant, animal, yeast and in some cases the properties of
the particular message you want to assay.  Roy Parker's lab has done lots
of work with yeast, including looking at poly(A)-tail length.  Pam Green's
lab has done lots of work with plant mRNA stability.  There are lot's of
people who work on mRNA stability in mammalian cells.  There's no one
protocol.  I think your best bet is to go to the literature and find
something similar to what you are interested in doing.

By the way, in my opinion, the best experiments with mRNA stability involve
the use of regulated promoters, whereby transcription of the message of
interest can be shut off while minimally perterbing the cells.  Following
the transcriptional shut-off, mRNA levels of the message of interest can be
followed over time by northern blotting, nuclease protection, etc.  The
results of experiments with transcriptional inhibitors (such as
ActinomycinD), while useful and informative,  have to be interpreted with
caution.  Examples of regulated promoters are Gal1 in yeast, c-fos in
mammalian cells, and a number of tetracyline regulated promoters which have
been developed for mammalian and plant systems.  I'm sure there are others,
as well.


Mike


Michael L. Sullivan, Ph.D

Institute for Molecular Virology
University of Wisconsin-Madison
1525 Linden Drive
Madison WI, 53706

(608) 262-3035 Phone
(608) 265-9214 FAX


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