RNase Protection Assay

Antonin Tutter atutter at jeeves.ucsd.edu
Sun Feb 23 23:05:15 EST 1997

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

More information about the Methods mailing list