A slot blot adaptation for analysis of RNASE protection assays

The ribonuclease protection assay (RPA) is commonly used to quantify levels of rare mRNAs in the cell. However, the polyacrylamide gel electrophoresis (PAGE) step in the RPA procedure is time-consuming and hazardous. This stu dy reports the development of an adaptation for the RPA which allows accura te quantitative analysis of large numbers of mRNA samples by replacing PAGE with direct application of the digested sample onto the hybridization memb rane by way of a slot blot. To test this protocol, three experiments of thr ee runs each were performed using dilution series of 6 to 200 pg of interle ukin-2 sense RNA transcript. ANOVA produced P-values of <.0001 and r(2) val ues of 0.93, 0.98 and 0.93 for the three experiments, demonstrating that th e data sets were highly linear. This method has also been used to detect th e full range of expression of an interleukin-2-producing mouse cell line. T here has been one previously published slot blot/RPA protocol, but this met hod differs in that it preserves the basic advantages of RPA over northern blotting; i.e., greater sample capacity and hybridization efficiency in sol ution. As well, this method can be simply added onto any working RPA protoc ol.