dsRNA blots

Mike Rott mike.rott at tuebingen.mpg.de
Mon Nov 6 12:37:55 EST 2000



Actually, no, as ssRNA treated in this manner gives excellent results and
is a lot more unstable then dsRNA.


mike

In article <3A06E633.45F10FDF at gpu.srv.ualberta.ca>,
wgallin at gpu.srv.ualberta.ca wrote:

> RNA is subject to base catalyzed hydrolysis, courtesy of its vicinial
> diols.  I would guess that you are hydrolyzing most of your target
> during your denaturation and immobilization steps.
> 
> Warren Gallin
> 
> Juergen Oberstrass wrote:
> > 
> > In article <mike.rott-0511001446020001 at ppp285.urz.uni-heidelberg.de>,
> > mike.rott at tuebingen.mpg.de (Mike Rott) wrote:
> > 
> > > I am having problems with dsRNA blots. I use a nondenaturing TAE agarose
> > > gel. treat in 50mM NaOH for 30 min and then blot with 20xSSC to charged
> > > nylon membrane. Membrane is then treated for 10 min in 50mM NaOH to fix
> > > and denature. Using this method with either dsDNA or ssRNA results in
> > > excellent blots but not from dsRNA. I assume that 50mM NaOH is not
> > > sufficient to denature dsRNA and am going to try a higher concentration.
> > > In the meantime what have others tried that worked.
> > >
> > >
> > > mike
> > 
> > I had a similar problem. dsRNA is the most stable nucleic acid
> > configuration.
> > One alternative is to change the gel system to phosphate buffered
> > polyacrylamid gels which unfortunately need buffer circularization. But
> > then you can denature the gel in glyoxal prior blotting and transfer the
> > denatured RNA.
> > Alternatively you can run a denaturing gel. If you need the information
> > if the RNA was double stranded you hybridize subsequently with strand
> > specific probes.
> > 
> > Good luck, Juergen
> > 
> > --
> > Juergen Oberstrass               E-Mail: oberstra at hrz.uni-kassel.de
> > Kassel University Genetics Department
> > Heinrich Plett Str. 40
> > D-34132 Kassel / Germany






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