"reverse northern" problems

[eric anderson]

In article <32A7113B.989 at students.wisc.edu>, eheinecke at STUDENTS.WISC.EDU wrote:


Liz,

we recently had problems detecting rare differentially displayed genes on
Northerns because we lost some of the responsiveness in our cell line so
we had to come up with a way to get the signal better.  the solution was
basically using a bigger hammer.  instead of using 20ug of total RNA I
went up to about 50-75ug in each lane and tripled the amount of labeled
(32P-random primed) cDNA that I used to probe them.  this seems to work
pretty well for most of the genes.  so in your case, while i think that
you need to do the Northerns eventually i would try blotting lots more DNA
and/or increasing the amount of labelled probe that you use.

secondly, i found that the type of membrane you use can have a
difference.  i saw that, using the exact same protocol, two different
membranes gave dramatically different resolution.

good luck,

eric
> I am currently working with mRNA differential display.
> Having cut out and subcloned several possible differentially 
> displayed bands, I am attempting to confirm differential expression
> using the "reverse northern" technique.  I realize Northerns are the
> ultimate confirmation method, but I'm trying to develop this dot 
> blot technique as a quick screening method for candidate cDNAs.
> To make a long story short, I've tried dot blotting both mini-preps of
> my subcloned cDNAs and PCR-amplified cDNAs, followed by probing
> with 32P-labeled (random primed) cDNAs from 2 separate strains of the 
> organism we work with. Both the control for my probe and another
> control cDNA from the organism are showing up.  Unfortunately, I
> have yet to see anything hybridizing to the cDNAs of interest.
> I may try longer hyb. times, since these may be rare mRNAs. 
> Any other suggestions?
> 
> Liz Heinecke

-- 
Eric C. Anderson
Memorial Sloan-Kettering Cancer Center
Sloan-Kettering Institute
1275 York Ave. Box 470
New York, NY  10028
(212) 639-2977
e-anderson at ski.mskcc.org