( ...)RNase Protection Assay (RPA),,,Using DIG-Labeled RNA Probes

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 the sample by the appearance of an appropriately sized probe fragment.

Factors Influencing the Assay

Transcript quality

• 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 template.
• 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 5 overhang.
  
  

Quality of target RNA

• RNase contamination: When probe fragmentation is observed in the protection assay, check the integrity of your RNA sample by agarose electrophoresis.
• DNA contamination
  
  

Amount of sample and labeled probe

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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