nuclear run-on for low abundant RNA

Bernard Murray bernard at elsie.nci.nih.gov
Fri Jun 28 20:00:22 EST 1996


In article <cchughes-2806961320500001 at cchh.bio.uci.edu>, cchughes at uci.edu 
says...
>
>In article <1996Jun28.180353.17694 at alw.nih.gov>, bernard at elsie.nci.nih.gov
>(Bernard Murray) wrote:
>
>
>> 
>> An alternative is to look at the level of hnRNA for your transcript.
> (...as apposed to doing a run-off.)
>
>Whoa...hang-on there.  I haven't read the article yet but looking at the
>level of hnRNA measures the *combined* rates of transcription *and*
>processing. It is not necessarily a good substitute for a run-off as
>regulation of processing seems to be an important rate limiting step for
>some genes.  See for example some of the recent literature on regulation
>of processing and transcription of the IL-2 gene.  We developed a PCR
>method for comparing processed and non-processed RNA levels but could
>never convince ourselves that we were measuring transcription rate per se.
>
>Just something to bear in mind.

I agree entirely that the limitations shoule be acknowledged.  However,
nuclear run-on analysis also measures the net rate of hnRNA production
(or at least rarely distinguishes between immature and processed RNA) so I
think the same caution should be applied to both run-on and hnRNA/RT-PCR
analyses.  The ideal technique would assay the production of new mature
RNA transcripts but I don't know of any easy way of doing this.  My
naiive view is to look at the "steady state" RNA levels and compare them
with the "rate" of transcription by run-on (or hnRNA) analysis.  If the
two are proportional it is reasonable to assume that transcriptional
activity governs the change in [mRNA] and if they are not proportional the
RNA processing/stabilisation/degradation plays a role.
	If you have evidence that the output (eg. slot blotted) from a
nuclear run-on reflects only mature RNA transcripts I would be very
interested to hear about it.  Your PCR method sounds like a good way to
compare the processes.

		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)
"But, keep in mind, I'm intensely stupid." -Crow T. Robot, MST3k
					(Attack Of The Giant Leeches)







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