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Ribonuclease Protection

  • Joanne L. Thorvaldsen
  • Marisa S. Bartolomei
Part of the Methods in Molecular Biology™ book series (MIMB, volume 181)

Abstract

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.

Keywords

Imprint Gene Hybridization Buffer Microfuge Tube Ribonuclease Protection Assay Deionized Formamide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Joanne L. Thorvaldsen
    • 1
  • Marisa S. Bartolomei
    • 1
  1. 1.Howard Hughes Medical Institute and Department of Cell and Developmental BiologyUniversity of Pennsylvania School of MedicinePhiladelphia

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