Northern blotting

Alain Nepveu Alain at
Wed Jul 26 10:40:56 EST 1995


In article <aquilla.1156985752C at sadye.emba.uvm.edu>,
aquilla at salus.med.uvm.edu (Tracy Aquilla) wrote:

> In Article <3urpas$54e at gazette.bcm.tmc.edu>, mtuvin at bcm.tmc.edu (Michael J.
> Tuvin) wrote:
> >In article <3ue8nn$f8l at azure.acsu.buffalo.edu> v115t2vq at ubvms.cc.buffalo.edu
> writes:
> >>A straight and simple question:
> >>Is there any probe better than rRNA/actins in Northern blot for internal 
> >>control?
> >>Thank you!
> >>Larry Lu
> >>Dept. of Biochem. Pharmac.
> >>SUNY at Buffalo
> >>V115T2VQ at ubvms.cc.buffalo.edu
> 
> >Sure. cRNA/GAPDH or cDNA/GAPDH. Glyceraldehyde 3-Phosphate Dehydrogenase is
> >less abundant and more uniform then actin.
> >We by our probe from Clontech and very happy with it. 
> >---------------------------------------------------------------------------
> >Michael J. Tuvim 
> >Research Assistant Professor, Dept.of Medicine, Baylor College of Medicine.
> >Tel: 713/794-7794(5), FAX: 713/794-7853, e-mail: mtuvin at bcm.tmc.edu
> >---------------------------------------------------------------------------
> 
> I'd suggest using a rRNA as the internal
> standard, even if it's a pain. 
>     tracy

I agree that ribosomal RNA is probably the best control.  One additional
reason for this is when the mRNA under investigation is of large M.W.
(higher than 4 Kb). In my experience, and for reasons I ignore, larger mRNA
is often degraded faster than short RNA of ~ 1Kb.  This can be seen by
comparing the 28s and 18s ribosomal RNA after staining with ethidium
bromide: the 28s RNA band sometimes disappears while the 18s band appears
intact.  Because of this, as a control, we always run an agarose gel in
parallel and keep the picture.  We still include an actin or GAPDH probe in
our RNase mapping hybridization mix because it provides an internal
control, but it has the limitations that were listed above.  



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