RNAse protection assay

[Emmanuel Skoufos, Ph.D.]

In article <cjNA4qK00iV847fqZq at andrew.cmu.edu>, Katherine Schaefer <ks3f+ at andrew.cmu.edu> writes:
>    I am trying to measure the kinetics of RNA:RNA pairing using an
>RNAse protection assay.  Briefly:
>    I initiate the pairing reaction (one 5' end-labelled RNA, one
>unlabelled RNA).  At time points, I take an aliquot of the pairing
>reaction and digest for 15 sec with a large amount (10ug/ml) of RNAse A.
> Reactions are stopped with phenol/chloroform, and the reaction is
>separated on a sequencing gel. 
>     The problem:  Various time points totally disappear!  For instance,
>the 1 min. time point has 50% of the RNA protected, the 1.5 min. time
>point has none of the RNA protected, and then the 2 min. time point has
>75% of the RNA protected. The points that disappear vary from reaction
>to reaction. 
>    Any thoughts?
>    Thanks,
>       Katherine Schaefer
>       Dept. Biology, Carnegie-Mellon Univ.
>       Email ks3f@ andrew.cmu.edu 

It seems that you are using way too much RNAse A.  Try to find the optimum
concentration of RNAse for your experiment.  The concentration that I use
when I do RNAse protection experiments (DNA:RNA hybrids,though) is 25 ng/ml.

I hope this helps

Emmanuel





_______________________________________________________________________________

Emmanuel Skoufos, Ph.D.
Insect Molecular Biology Group
Institute of Molecular Biology and Biotechnology
Box 1527 
Heraklion, Crete 71110
Greece