Quantitation of RNA by ribonuclease protection assay.
A robust ribonuclease protection assay is described here. In brief, total cellular RNA, carrier yeast transfer RNA, and 32P-labeled antisense riboprobes, (one or more designed to detect the RNA species being studied and another to detect a suitable RNA species to act as a loading control) are combined and made 0.5 M with respect to ammonium acetate. Absolute alcohol (2.5 vol) is added, and tubes are incubated at -20 degrees C for 30 min. Precipitated RNA and riboprobes are pelleted by centrifugation and, after removal of the supernatants, dissolved in a small volume of hybridization buffer. After hybridization for 16 h at 55 degrees C, a ribonuclease cocktail is added to digest the unhybridized RNA. This is followed by a proteinase K digestion step that degrades the ribonucleases. Finally, the hybridized complex is precipitated at -20 degrees C using isopropanol:4 M guanidium thiocyanate (2:1), with added glycogen as a coprecipitant, and harvested by centrifugation. The pellet is dissolved in loading buffer, and the sample is electrophoresed in a polyacrylamide gel. The intensities of the bands in the gel representing the protected fragments for the target RNA and the loading control are quantitated by phosphorimager analysis.
Molecular Oncology Laboratories, Cancer Research UK, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford.
John W
JW
Moore
D001681:Biological Assay / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D015347:RNA Probes / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*
D015347:RNA Probes; D012313:RNA; D012260:Ribonucleases