RNAse Protection Assay

kim at m44.unm.edu kim at m44.unm.edu
Thu Feb 6 14:56:59 EST 1992


Hello Group:

I am thinking about the utility of RNAse protection assays for detecting and
quantitating rare transcripts from total RNA samples.  I saw a paper in
Analytical Biochemistry (Thompson and Gillespie. 163:281-291. 1987) in which an
RNA probe is hybridized with target RNA in a crude guanidine thiocyanate cell
lysate.  After dilution, the hybrids are digested with RNAse and precipitated
with TCA onto a glass filter, then counted.

I have seen autoradiograms of S1 nuclease and RNAse protection assays, and
there is always considerable radioactive material in the gel besides the
protected probe.  What is this stuff?  If one counted all of the TCA
precipitable material in such a reaction, wouldn't this stuff give a spuriously
high count?  Does the low mw radioactive material represent incompletely
digested ss probe, or is it over-digested "protected" probe?

I very much dislike having to compare relative signal by looking at an
autoradiogram, and I don't think that formalizing the process by using a
scanner really satisfies the requirements for a quantitative measurement.  I
would much rather digest all the ss stuff away and count precipitable material
remaining by LSC.  Is this a bad idea?  

Thanks for listening.

Daniel Kim   KIM at FLOVAX.LANL.GOV



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