RNase Protection Assay

Marieke R. Koedood Zhao rkoedood at bio.bu.edu
Thu Feb 27 12:27:16 EST 1997


We found that hybridization temp is the place to start. We would typically
hybridize only a few degrees (eg. 5 degrees) below the melting temp.

Marieke

Antonin Tutter (atutter at jeeves.ucsd.edu) wrote:
: mennow at ruly46.medfac.leidenuniv.nl wrote:
: > 
: > I'am trying get get an RPA working for several messengers.
: > So far I've had bands with loads of background with some probes and no bands with other probes.
: > What are the best ways to optimize RPA.
: > Start varying with hyb. temperature, vary the amount of RNase A and Rnase T1, vary the amount of probe added to the RNA,
: > increase specific activity of the probes or just switch to an other region for a new probe?
: > Please suggestions.
: > 



: I had the same bad luck trying to use nuclease S1 protection to
: quantitate a message.  What you mentioned is true -- all those
: parameters need to be optimized before you get nice results.  I also
: found that the design of the primer is **critical** -- make sure there
: are little or no self-homologous regions or stem-loops that form on your
: primers.  This can affect not only how efficiently the unbound primers
: get degraded, but also this can affect how well the primers anneal to
: the message.

: Best of luck,

: Anton Tutter
: Salk Institute
: atutter at aim.salk.edu



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