The ribonuclease protection assay (RPA) is a highly sensitive
and specific method for the detection and quantitation
of mRNA species. The assay was made possible
by the discovery and characterization of bacteriophageencoded
DNA-dependent RNA polymerases (SP6, T7,
and T3). These enzymes possess several properties that
make them well suited for the preparation of highly specific
hybridization probes. They are very specific in their
action, recognizing only phage-specific promoter
sequences that are not part of other DNA sequences.
To generate a probe for use in this assay, it is necessary
to subclone a fragment that contains the cDNA
sequences of interest downstream of one of the mentioned
phage promoters. The orientation of the insert
must direct the synthesis of complementary (anti-sense)
RNA. Ideally, this construct should be digested with a
restriction enzyme (RE), which produces 5 overhangs, to
create a linear template that will be transcribed into a
100 to 300-base run-off transcript.
The RPA technique is based on the hybridization in solution
of a labeled anti-sense probe RNA with the RNA
pool being tested. Complementary portions of labeled
probe and mRNA from the pool to be analyzed form
hybrids that are resistant to digestion with single strandspecific
RNase T1 and RNase A. Because of its sensitivity,
the RPA can, in general, be performed on total RNA
preparations derived by standard methods from either
frozen tissues or cultured cells, without further purification
of polyA+ RNA.
Following treatment with ribonuclease, the RNase-protected
fragments are resolved on a denaturing polyacrylamide
or agarose gel. Subsequent detection reveals
the presence of target mRNA in t
he sample by the
appearance of an appropriately sized probe fragment.
Factors Influencing the Assay
Quality of target RNA
- Long transcripts tend to give incomplete transcribed
probes. We suggest the use of a relatively small probe
for this assay (100300 nucleotides).
- RNase contamination
- Presence of transcription-termination signals in the
- Wraparound transcripts resulting from the use of REs
that produce either a 3 overhang or a blunt end. It is
strongly recommended to use a RE that produces a
Amount of sample and labeled probe
- RNase contamination: When probe fragmentation is
observed in the protection assay, check the integrity
of your RNA sample by agarose electrophoresis.
- DNA contamination
The sample size is usually in the milligram-range and the
amount of labeled probe is around 500 pg1,000 pg.
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