"reverse northern" problems
Gregory S. Buzard 'Buz'
buzardg at MAIL.NCIFCRF.GOV
Thu Dec 5 19:47:45 EST 1996
>..currently working with mRNA differential display. Having cut out and subcloned several possible differentially displayed bands, a=
m attempting to confirm differential expression using the "reverse northern" technique.
(Actually, what is described is the reverse blot
>..realize Northerns are the ultimate confirmation method, but I'm trying to develop this dot blot technique as a quick screening me=
thod for candidate cDNAs. tried dot blotting both mini-preps of subcloned cDNAs and PCR-amplified cDNAs, followed by probing with 32=
P-labeled (random primed) cDNAs from 2 separate strains of the organism we work with. Both the control for probe and another control=
cDNA from the organism are showing up. Unfortunately, yet to see anything hybridizing to the cDNAs of interest. may try longer hyb.=
times, since these may be rare mRNAs. Liz Heinecke U.W. Madison.
To start with, did you generate the cDNA for the initial
Differential Display PCR with an oligo-dT primer? If so,
then the same technique should be used for generating your
labeled probe total cDNA. If you are random priming, you
might be SELECTING! (bad-bad) for longer or shorter cDNA
Did you do an initial subtraction step to simpify your cDNA
pool for template-unique transcripts?
Both your probe control and your control cDNA may be too
highly represented on the blot to allow a fair search for
differentially expressed rare copy mRNAs. Try diluting them
10,000-fold, and then reprobing with labeled cDNA. Longer
hybridizations are probably not going to get you where you
want to go, unless you have been doing it wrong at too high
Then there is the high probability, from what others are
posting, that your clones are false positives.
More information about the Methods