The ribonuclease protection assay (RPA) is a sensitive technique for the analysis of total cellular RNA. It involves generating a specific antisense riboprobe, hybridizing the probe to total RNA, removing unprotected RNA by RNases, and finally isolating and analyzing the protected RNA on a denaturing gel. Although the RPA is somewhat more labor-intensive than Northern analysis, it has the advantage of being more sensitive (as little as 0.1 pg of target RNA can be detected with ideal hybridization conditions). RPAs are also more tolerant of partially degraded RNA (provided the area that is protected is intact). Although RPAs are not as sensitive as polymerase chain reaction (PCR)-based RNA analyses, the target RNA is analyzed directly; a reverse transcription step is not required. Finally, the RPA is quantitative as long as the probe is in excess. More important for the study of imprinted genes, the RPA can be designed to detect allele-specific expression of the target gene of interest.
KeywordsImprint Gene Hybridization Buffer Microfuge Tube Ribonuclease Protection Assay Deionized Formamide
- 2.Gilman M. (1993) Ribonuclease protection assay, in Current Protocols in Molecular Biology (Ausubel, F. M., et al., eds.), Wiley, New York, pp. 4.7.1–4.7.8.Google Scholar
- 3.Belin D. (1998) The use of RNA probes for the analysis of gene expression, in Methods in Molecular Biology, Vol. 86 (Rapley, R. & Manning, D. L., eds.), Humana, Totowa, NJ, pp. 87–102.Google Scholar
- 8.Leighton P. A., Saam J. R., Ingram R. S., Stewart C. L., and Tilghman S. M. (1995) An enhancer deletion affects both H19 and Igf2 expression. Genes Dev. 2079–2089.Google Scholar
- 14.Sambrook J., Fritsch E. F., and Maniatis T. (1989) Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar