reverse northern?

Andy Phillips andy.phillips at bbsrc.ac.uk
Thu Oct 23 08:23:02 EST 1997


Harold E Smith wrote:
> 
> I would like to probe a blot of cDNA clones with mRNA isolated from two
> different tissues to identify clones that are differentially expressed.
> I'm fairly certain it's been done before, but I can't find a reference.
> Would someone be so kind as to point me in the right direction?
> Thanks in advance,
> Harold Smith

This has two problems: firstly, abundant mRNAs label up to a very high
level and and are represented by a large proportion of clones in the
library. These will tend to mask faint signals from less abundant mRNAs,
unless you plate the library at low density. Secondly, the less abundant
mRNAs will label to only a very low level and you'll have trouble
detecting a signal from an mRNA of less than, say, 0.01% abundance. So
if the clones you expect to find are abundant, OK; if they're rare,
forget it. 

I would also ignore recipes that label the mRNA by reverse transcription
- most recipes include some cold dNTPs so you get a relatively cold
probe. I made first strand cDNA first (I used a home made recipe but
there are plenty of kits these days) than labelled this by an
oligolabelling method (kit again if you like).

Generally speaking, differential hybridization has been superceded by
more sensitive methods such as subtraction and differential display.
These are, however, technically challenging and I would try diff. hyb.
first - you may be lucky.

Andy


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