mRNA Transcript Quantitation Method [Q]

Ned Mantei mantei at neuro.biol.ethz.ch
Tue Nov 5 01:27:37 EST 1996


In article <mnoda-0111961246560001 at 207.126.101.77>, mnoda at lipovx.lbl.gov
(Michael Oda) wrote:


>      We are looking for a means to quantitate a transcript from
> mouse liver. Under the conditions of the experiment we expect a
> percent change in levels of transcript per mass of tissue in
> the 5% to 20%  range. So we are in need of a fairly accurate method
> of quantitating. I have heard that RNAse protection may be one
> of the better ways but I was wondering if there were others.
> Avoidance of radioactivity is also a major plus to any method 
> suggested.

You might try RNase protection or S1-mapping (with an end-labelled probe)
with probes extending over the 5'-end of the transcript. This way,
remaining undigested, full-length probe doesn't contribute to the signal.
You can include in every sample a small amount (say 0.01--0.1 fmol) of an
in vitro transcript shorter at the 5'-end, as an internal control for
losses during work-up and application of the sample to the gel. An
external standard, prepare an in vitro transcript extending from more or
less the 5'-end of the natural transcript to a point past the 5'-end of
the probe; isolate this transcript on an agarose gel and quantitate by UV
absorbance. Use varying amounts in a series of standards to see how small
a difference you can consistently detect.
However, my experience has been that anything under about 2-fold
difference is not very reliable. I doubt that one can repeatedly detect
5--20% differences.

-- 
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046



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