nuclear run-on for low abundant RNA

Bernard Murray bernard at elsie.nci.nih.gov
Fri Jun 28 13:03:53 EST 1996


In article <4r10h4$78r at newz.oit.unc.edu>, mahayden at med.unc.edu says...
>
>     I'm currently setting up the nuclear runoff assay in our lab.  
>You're right that it requires a lot of 32-P, but there is no getting 
>around that- chemiluminescent methods don't work with this assay.
[SNIP]

Maybe they do in some people's hands.  Check out Merscher et al. (1994)
"Nuclear runoff transcription analysis using chemiluminescent detection"
Biotechniques 16, 1024-1026

I haven't tried this personally but it looks very nice.  It takes
20 - 50 million nuclei and uses DIG UTP.  With the latest high sensitivity
AP substrates you may be able to use less nuclei or look at lower
transcription rates.

An alternative is to look at the level of hnRNA for your transcript.
In this case you don't have to extract nuclei, just RNA.  The level
of immature unprocessed RNA (by RT-PCR) gives a good reflection of the
rate of transcription.  A version of this technique was recently
published by Elferink & Reiners (Wayne State U.) in Biotechniques
(sorry, I don't have the actual reference available).  This may need
10-fold less cells than a run-on.
	Anything to stay cool...

		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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