RT-PCR vs. RNase protection assay

Rae Nishi nishir at ohsu.edu
Wed Jun 19 14:20:57 EST 1996


We use both RT-PCR and RPAs in my lab, so I can comment on how the two
compare.  Basically, I trust RPA more than PCR because we have run into
some amazing PCR artifacts (also we have had problems with our favorite
transcripts contaminating reactions).  The quantitative PCR we do is a
competitive PCR using a competitive template that we make that
generates a different size product with the same primers.  I like RPA
because you can get more information out of a single aliquot (usually
20 ug) of total RNA--we typically run multiprobe assays-- 2 to 3
transcripts of interest plus a constitutive, hopefully non-regulated
control transcript.  However, RPA is not as sensitive as RT-PCR-  so
when we want some quantitative info about transcripts from very small
tissues (eg., embryonic tissues) or primary cell culture-- we have to
go to competitive PCR.  I tend to think of PCR and RPA as being roughly
comparable in cost.  The enzymes for both are rather expensive.  RPA is
more labor-intensive because we have to gel purify our probes and it
takes time to pour/dry the polyacrylamide gels that you run your
digests on.

Rae Nishi, PhD
Associate Professor
Dept. Cell & Developmental Biology
Oregon Health Sciences University
Portland Oregon 97201
**that's Orygun, NOT Ora-Gone**



More information about the Methods mailing list