RNAse protection assay?

tronni at nphi.fi tronni at nphi.fi
Thu Jul 22 13:55:27 EST 1993


Hello all bio-netters!

I'm studying the expression of some cytokine genes in human
mononuclear cells and have found out that the sensitivity
of Northern blotting is not always enough with total RNA
(even with 20 microgram samples per lane).
Isolation of mRNA is out of the question (too low yields).

I'm considering the "RNAse protection assay", where you
hybridize 32P-labeled antisense-RNA probes with target
mRNAs in solution. I've read from many sources that 
it should be more sensitive than Northern.

Recently I saw in Biotechniques an advertisement by Ambion about their
kit, which allows the reaction to take place in a single
tube and you can even use multiple probes to detect many mRNAs
simultaneously from total RNA.

I would be grateful for any hints and opinions about
this. Should I get a kit or build my own?
Is this assay really as good as advertised? Do the signals
get too hot to be comfortable to work with?

Cheers,


Tapani Ronni
National Public Health Institute
Mannerheimintie 166
00300 Helsinki
FINLAND 



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