How can I tell if RNA is degraded?

Vellanoweth's Lab jwheele at calstatela.edu
Wed Apr 26 20:49:44 EST 2000


Hi Paul, thanks for your input.  How can I tell, other than a
visualizing agarose gel w/EtBr, if the RNA is degraded?  The RNA looks
beautiful on the qualitative gel, 2 tight bands sometimes a few below,
all lanes migrating the same etc. (gel is 1% agarose w/ TAE, no
formaldehyde).   However, someone suggested to me that the RNA might be
degraded and migrating together conformationally, then when the
formaldehyde hits it the RNA denatures and falls apart... I've never
heard of this before, how would one be able to tell if this were the
case?

No one has ever heard of formaldehyde degrading RNA so I'm stumped.
I've ordered a new bottle of formaldehyde and will test it with that one
as well.

In the diagnostic gels (no formaldehyde in gel, just 1% agarose + TAE)
the samples incubated at 65 C for 15 min with:
formamide (RNA suspension)  alone;
formamide + MOPS,
formamide + dye,
formamide + MOPS, dye
all ran perfectly with tight bands

samples with:
formamide + formaldehyde
formamide + MOPS + dye + formaldehyde
degraded completely.

thanks for any help...

Janel Wheeler
Vellanoweth Biochemistry Lab
Calif State University, Los Angeles




Paul Klosen wrote:

> Vellanoweth's Lab wrote:
> >
> > We've been having a lot of trouble degrading our plant RNA with
> > formaldehyde.  We've tried pHing the formaldehyde with NaOH to 5.3,
> and
> > eliminating the formaldehyde from the incubation step.  However we
> are
> > still getting degradation in the gel.  I looked in the Ambion
> catalog to
> > check if our formaldehyde was bio-reagent quality and found they
> don't
> > sell formaldehyde, only formamide.  This leads me to wonder whether
> > formamide can be used in a denaturing gel rather than formaldehyde,
> or
> > if you can run it without the denaturant since the sample is already
> in
> > formamide.  Any northern experts out there?
>
> Formaldehyde is a potent inactivator of almost all enzymes, because it
>
> reacts covalently with proteins. It even inactivates RNAses. If you
> have
> trouble with degraded RNA in your gels, the RNA probably is already
> degraded before it got into your gel. Are you certain about the
> integrity of your RNA ?? Maybe you are dealing with incomplete
> denaturation of you RNA before electrophoresis ??
>
> --
> Paul Klosen, PhD
> CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
> Universite Louis Pasteur  12, rue de l'Universite F-67000 Strasbourg,
> FRANCE
> Tel. 03.88.35.85.04  Fax. 03.88.24.04.61
> klosen at neurochem.u-strasbg.fr





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