RNase Protection Assay: Non-specific?

Rolands G. Aravindan aravind at wsunix.wsu.edu
Sat Feb 21 00:43:10 EST 1998


Hello friends,

I have never actually done RPA, but a Professor in the department showed
me a RPA blot that had just returned from a journal after initial review.
The reviewer's criticism of the blot was rather strange to me. I wonder
how one could explain/rebutt it.

Let me explain the blot, in short. It is rather clean with clearly 
treatment dose-dependent increase in the expression of POMC mRNA and the
standard was constant. Both signals are remarkably strong and the
background is remarkably white and clean; except for another minor (about
1/100 the specific band) band, also appearing dose-dependent, about 100
bases lesser in size. Considering the strength of the specific signal, the
blot could have been sent in with slightly decreased exposure to film
without compromising authenicity (?; wrong?) in the first place.

Strangely, the reviewer has asked "whether the minor POMC band was also
scanned"! Being quite familiar with Northerns and the like, I would have
thought that there really is no confusion as to the fact that the minor
band is not related to POMC and that it is nonspecific. Or am I wrong?
Anybody out there have any suggestions as to how one could explain/rebutt
the reviewer's comment?

I would be very grateful for all replies, either directly or via the NG.

Thanks in advance,

Sincerely,

Aravind
aravind at vetmed.wsu.edu




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