RNase protection assay

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The ribonuclease protection assay (RPA) is a highly sensitive and specific method for the detection of mRNA species. The assay was made possible by the discovery and characterization of DNA-dependant RNA polymerases from the bacteriophages SP6, T7 and T3, and the elucidation of their cognate promoter sequences. These polymerases are ideal for the synthesis of high-specific-activity RNA probes from DNA templates because these polymerases exhibit a high degree of fidelity for their promoters, polymerize RNA at a very high rate, efficiently transcribe long segments, and do not require high concentrations of rNTPs. Thus a cDNA fragment of interest can be subcloned into a plasmid that contains bacteriophage promoters, and the construct can then be used as a template for synthesis or radiolabeled anti-sense RNA probes.

Ribonuclease protection assay is used to quantitate mRNAs. The method is based on the hybridization of a target RNA to either a labeled anti-sense RNA probe in vitro transcribed from a DNA template. RNase treatment follows, resulting in degradation of singlestranded RNA and excess probe. Double-stranded RNA is not degraded by RNASE treatment. The probe and target RNA are resolved by denaturing polyacrylamide gel electrophoresis and the "protected" probe is visualized using autoradiography or beta imaging equipment.