18S and 28S in Northern blot
wgallin at gpu.srv.ualberta.ca
Wed Jun 14 13:06:35 EST 2000
Nick Theodorakis wrote:
> Mano <mdep at musica.mcgill.ca> wrote:
> >The mRNA of interest migrates with the 18S rRNA..... I am
> concerned that
> >the signal I see is in part contributed by the 18S rRNA....
> however, I
> >have no other signal and no reaction from the 28S rRNA... is it
> more or
> >less safe and not to worry since the 28S rRNA does not cross-
> react. In
> >other words, in the case of cross-reactivity with the 18S rRNA,
> >one expect to see it with the 28S rRNA also??? I guess I
> should just
> >run polyA guess to make sure.. comments are very welcome.
> No, I've probes that have cross-hybridized with one or the other
> rRNA but not both.
> What I do is stain the blot with methylene blue. Then overlay
> the autoradiogram with the blot (or a photocopy of the stained
> blot) to see if the signal exactly matches the rRNA staining
> pattern. (You may need to mark the blot and exposure with a glow-
> in-the-dark sticker to line them up). Even if an actual mRNA is
> the same size as the rRNA, it is usually distinguishable from it
> (e.g., it might have a thinner band). If it exactly overlays it,
> I would strongly suspect cross-hybridization.
If your signal is coming from cross-hybridization with 18S rRNA, then it
will appear at equal levels in RNA isolated from all tissues (assuming
that you have loaded equal amounts of RNA on all wells). If it is
recognizing a specific message, you would expect to see variable signal
levels in RNA isolated from different tissues, and it would be
reasonable to expect to see at least one tissue where the mRNA is not
expressed at all and therefore would give no signal.
Also, if you compare polyA-plus RNA to total RNA, your hybridization
should be much stronger in the polyA-plus well, if the lanes are loaded
with equal amounts of RNA.
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