A robust ribonuclease protection assay is described here. In brief, total cellular RNA, carrier yeast transfer RNA, and 32 P-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 0.5Mwith respect to ammonium acetate. Absolute alcohol (2.5 vol) is added, and tubes are incubated at −20�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�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�C using isopropanol:4Mguanidium 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.
