Northerns...AGAIN!

Dom Spinella dspinella at chugaibio.com
Mon Oct 6 11:09:29 EST 1997


> Can any one give me a cluse as to what to do? I started working with RNA
> and Northern blots last September. When I finally went to do a Northern,
> it worked. However, that was the first and only time. The lab tech and I
> have been struggling with it ever since. For the longest time we had a
> problem with black blots which turned out to be a problem with the
> membrane we were using. We have since swithced to HYBOND and while the
> control probe for the amount of RNA loaded works fine, the other probe I
> am using isn't working.
> 
> My supervisor and the lab tech have been using this probe for a long time.
> My supervisor did all her thesis work with this. However, the problem now
> is that we make the probe and hybridize. The counts seem to be quite
> high when the counter is placed next to it after hybrizidation. The
> problem is that the wash solution cannt go near the membrane and all the
> probe literally disappears. My supervisor also told me this past week
> that I have non-specific binding. The probe is still the same as my
> supervisor's but even with the same wash solution, the probe is not
> working as it should be. It isn't working at all!
> 
> The wash solution used was:
> 
> 1X SSC,1%SDS 15 min(RT)
> 0.5 X SSC,0.5%SDS 15 min(RT)
> 0.1 XSSC,0.15 % SDS 2 X15 min (RT)
> 0.1 X SSC 0.15%SDS 30 min @RT
> 
> We can't get past the first 15 min wash now and the probe is literally
> gone.
> 
> As for the non-specific binding, I have no idea what to be doing for that
> either.
> 
> Any suggestions? I am getting desparate. I have been here a year and am
> now well into my second. My masters is only a two year project. Please,
> any info would be greatly appreciated.
> 
> Thanks,
> Joy :)

First, are you sure that your Northern is truly not working?  How
certain are you that the target message is actually present in your RNA
prep? You can always try backing of your stringency (e.g start with 2X
or higher SSC).  BTW, how is your RNA prep? Are you using total RNA or
mRNA?  Have you looked at it by direct staining of the gel, and do you
have much degradation? Second, what sort of probe is this (cRNA? DNA?)? 
What is its specific activity?  Are you sure it has even been labeled? 
Third, what are your hybridization conditions?  Are you sure that your
probe hybridizes to its target uner those conditions?  You could try
serially diluting a cloned cDNA (or better, cRNA) and ensuring that your
probe and hybridization conditions work by simple dot blots.

It doesn't sound to me like you have any non-specific binding problems. 
Your problem is too little binding, not too much.  All blots are hot by
Geiger right after they come out of the hybridization -- that is not
indicative of non-specific binding.

Sounds to me like a little more assay development work needs to be
done.  That's what Master's Thesis work is all about!  Good luck.  --
Dom Spinella



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