nuclear vs. total RNA isolation question

lsalati at wvnvms.wvnet.edu lsalati at wvnvms.wvnet.edu
Mon Jul 17 09:58:50 EST 1995


Hello,

I have a question about determining mRNA abundance in nuclear and
cytoplasmic fractions.  Note that I'm specifically interested in
mature message so intron probes would not be useful.

We would like to measure the abundance of our mRNA in the nuclear
and cytoplasm under different treatment conditions.  Our strategy
is to isolate nuclei using the citric acid procedure and measure
the mRNA differences between treatments by Northern blot.

Then for comparison run a Northern on total RNA.  There will be
some contamination of the cytoplasm with the nuclear fraction and
we would have to take that into account.  We thought that trying to
isolate the cytoplasmic fraction might be too difficult.

The question is:  How do you know that the mRNA you have detected
in the nuclear fraction is not substantially contaminated with mRNA
from the cytoplasm?  It is suspected that RNA/ribosomes bound to ER
can copurify with nuclei preps and thereby confound the results.

Is there a control RNA (of mouse or rat origin since that is our
system) one could use to determine if the nuclei were contaminated
with cytoplasmic RNA?

We thought about running the nuclei through multiple sucrose cushions
but in our experience even though you get cleaner nuclei you lose
a lot too.  In addition unlike for run-on assays we have to worry
about RNA degradation (we're working with liver where there are lots
of RNases).

Any suggestions?

Steve




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