Ribonucleas Protection Assay question

Mike Amstutz amstutzm at musc.edu
Fri Oct 24 10:04:40 EST 1997


Has anyone ever performed a ribonuclease protection assay in which the
"labeled" element was intracellularly derived mRNA? (as opposed to a
labeled probe mixed with harvested RNA?

My problem is that I'm tyring to measure the half-life of an mRNA which 
contains a destabilization element.  mRNA containing this
destabilization element has been shown to be stabilized when measured
using transcriptional inhibitors such as actinomycin-D and DRB.
Also I want to measure the half-life in my particular system (cell type,
vector etc) which so far does not appear amenable to transient
stimulation as some other systems (c-fos).

Therefore, I am thinking about doing a pulse-chase experiment to 
radiolabel intracellular mRNA, harvest this labeled-mRNA and use it
in a ribonuclease protection assay.  Does this sound feasible?
Any other suggestions?

Thanks 
amstutzm@@musc.edu
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