atutter at aim.salk.edu
Sat Jun 15 22:54:04 EST 1996
The most quantitative method is probably RPA (Ribonuclease Protection Assay). I am
currently using this method to quantitate messages transcribed from in vitro-reconstitued
chromatin templates. I contemplated using northerns or slot blots, but both are inherently
finicky, and RPAs are faster. I make 5' labeled dsDNA probes to my message of interest,
with substantial amount of probe (>100 bp) extending upstream of the start site. This
provides for a control (uncleaved probe). When I digest the annealed probes with S1, I get
a smaller labelled fragment. The only hitch is that the annealing conditions must be worked
out precisely, otherwise the probe gets digested and quantitation goes out the window.
Hope this helps,
BTW, if you find another method that seems to work, let me know!
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