"reverse northern" problems

Gregory S. Buzard 'Buz' buzardg at MAIL.NCIFCRF.GOV
Thu Dec 5 19:47:45 EST 1996


Liz wrote:

>..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 
technique)

>..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 
transcripts?

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 
a stringency.

Then there is the high probability, from what others are 
posting, that your clones are false positives.
Buz





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