northerns and pre mRNA

Bernard P. Murray, PhD bpmurray*STUFFER* at
Wed Sep 16 18:16:09 EST 1998

In article <6to9oo$2uo at>, Ruth McMahon
<Ruth.McMahon at> wrote:

> When doing Northerns, is there an assumption that only mRNA will be
> detected (when probing with a cDNA probe to total RNA)? If you have pre
> mRNA with introns small enough, or even partially spliced pre mRNA, such
> that it can still be picked up with your probe, is there a reason why
> this does not happen, or does it happen? (Is pre mRNA/partially spliced
> mRNA too transient in all situations?)
> Thanks for any clarification,
> Ruth.

You can certainly pick up unspliced mRNA with RT-PCR so it is there
somewhere even if at low abundance.  Since these RNA species tend
to be more concentrated in the nucleus you may lose them if you use
an RNA purification method that relies on spinning out the nucleii
(eg. detergent lysis followed by phenol extraction).  Also, these
larger RNA molecules transfer less efficiently to membranes than
their smaller mature counterparts.
     On most occasions it would be difficult to differentiate
between specific hybridisation to a larger RNA and general
background but if the RNA is rapidly transcribed and the mature
mRNA is unstable this may shift the balance to a point where
a specific signal is seen.
     I hope that clarifies my state of ignorance :-)
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA

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