RNA gels - staining and denaturing

Stephen R. Lasky, Ph.D. Stephen_Lasky at brown.edu
Fri Jul 14 09:33:26 EST 1995


In article <1995Jul13.233353.11895 at emba.uvm.edu>, amarion at moose.uvm.edu
(Amy Marion) wrote:

> Dear netters;
> 
>         I need some advice on RNA gels.  I have made 3 attempts to run
> total RNA through glyoxal/DMSO gels and one attempt with a formamide gel.
> In all cases I have been unable to see the RNA after ethidium bromide
> staining.  Even the RNA markers do not show up.
> 
> Should RNA be stained with ethidium bromide at all?  (These gels will be
> used for Northerns.)

Theoretically, ethbr, an agent that intercollates between stacked bases,
should not stain denatured nucleic acids very well (if you stain a DNA gel
with ethbr and so that you can see the bands and then denature the DNA
with NaOH in preparation for Southern blotting, the staining goes away
very rapidly.  I found this out when I realized that I had forgotten to
put a ruler on the gel for reference when I took the picture.  The gel had
only been in base for about 3 minutes, but the staining was gone
already).  But both agarose gel systems you used allow enough secondary
structure for the ethbr to stain the RNA (empirically).  

> What is the proper procedure for staining RNA gels (either glyoxal or
> formamide) with ethidium bromide?

Despite what Maniatis says, I always pore my gels with ethbr in them so
that they stain while they are running, I have not noticed any decrease in
transfer of RNA's to membranes, and you can see and record the RNA
(particuarly the 18 and 28 S bands) on the membrane by UV illumination. 
Another way to stain your RNA is to put about 1 microgram of ethbr in the
RNA denaturation mix just before loading it on the gel.  This gives
slightly less staining but a cleaner looking gel since the excess ethbr
runs out of the top of the gel during electrophoresis.

> 
>         I have run the RNA through 0.1% SDS, 1.2% agarose gels.  After
> staining with EtBr all the RNA (including the RNA markers) are visible.
> Will the 0.1% SDS denature the RNA?  If yes, can the RNA from this gel be 
> transfered to nylon for a Northern?

0.1% SDS is not a very efficient denaturation reagent.  You should be able
to get the glyoxal or formaldehyde gels to work.


Hope that helps

SRLasky

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Stephen R. Lasky Ph.D.  Brown University/Roger Williams Medical Center
Landline: 401-456-6572   Fax: 401-456-6569  E-Mail: Stephen_Lasky at brown.edu
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