Ribonuclease protection
J L Thorvaldsen1, M S Bartolomei
1Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|July 5, 2003
Summary
The ribonuclease protection assay (RPA) offers sensitive detection of total cellular RNA, outperforming Northern analysis and directly analyzing RNA without reverse transcription. This method is quantitative and can detect allele-specific gene expression.
Area of Science:
- Molecular Biology
- Genetics
Background:
- The ribonuclease protection assay (RPA) is a sensitive method for analyzing total cellular RNA.
- It offers advantages over Northern analysis in terms of sensitivity and tolerance to RNA degradation.
Purpose of the Study:
- To detail the methodology and advantages of the ribonuclease protection assay (RPA) for RNA analysis.
- To highlight its utility in studying imprinted genes and allele-specific expression.
Main Methods:
- Generating a specific antisense riboprobe.
- Hybridizing the probe to total RNA.
- RNase digestion of unprotected RNA, followed by isolation and analysis of protected RNA on a denaturing gel.
Main Results:
- RPA demonstrates high sensitivity, detecting as little as 0.1 pg of target RNA.
- The assay is more tolerant of partially degraded RNA compared to other methods.
- RPA allows direct analysis of RNA, bypassing the need for reverse transcription.
- The method is quantitative when the probe is in excess.
Conclusions:
- RPA is a sensitive, quantitative, and versatile technique for total cellular RNA analysis.
- Its ability to detect allele-specific expression makes it particularly valuable for studying imprinted genes.
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