Nonspecific riboprobe detection of 28S Ribosomal RNA?
Fred Boyd, Ph.D.
fredboyd at bmec.micro.umn.edu
Tue Feb 1 10:54:14 EST 1994
In article <2i0ngo$l9i at net.usuhs.mil>, denton at usuhs.ucc.usuhs.nnmc.navy.mil
> In Article <2hvdb4$e4o at mserv1.dl.ac.uk>
> dmsander at mailhost.tcs.tulane.edu (David M. Sander) writes:
> >Can anyone think of a reason why any particular riboprobe (32-P, 780 bases)
> >might show cross-reactivity with the 28S ribosomal band in a northern
> >blot/hybridization of total rat RNA?
> I have broad experience in this department. Unfortunately, it is
> an extremely common and annoying problem particularly with probes with
> GC compositions > 60%. While there is no other solution for the
> blots other than the one that you have already deduced, (I'm certain
> that you have tried the obvious-increasing the hyb. stringency and
> formamide concentrations) you might consider trying an RNase protection
> assay, if detection of the messaage is all that you require. I have never
> had any problem with these probes and GC content in such an analysis.
We have recently cloned a bit of DNA with several regions of homology to
28S. One regions is 51/53 nt identity. In reading some limited reviews of
28S DNA, the genes are described as "recombigenic," which I extrapolate to
suggest that 28S rRNA may recombine with a variety of other GC rich cDNAs.
Fred Boyd Voice- 612-624-8150
Lab. Med. Path. Fax- 612-616-2444
Cell Biol. Neuro. Lab- 612-624-8154
Box 609, UMHC
University of Minnesota E-Mail fredboyd at bmec.micro.umn.edu
Minneapolis, MN 55455
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