18S and 28S in Northern blot

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Tue Jun 13 23:28:23 EST 2000


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,
would
>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.
>Thanks.
>Mano
>
>
>

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.

Nick



Nick Theodorakis

nicholas_theodorakis at urmc.rochester.edu
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