Modifications of RNase protection assay for neuroscience applications

This report describes how certain modifications of the Ribonuclease (RNase) protection assay may increase its efficiency by decreasing the time and cost of the procedure without compromising reliability. We show that, under the experimental conditions tested, the RNA samples can be precipitated by a solution of Tri Reagent in ethanol immediately following the RNase digestion step. Drying the samples under vacuum before dissolving them in the gel loading buffer improves the consistency of the assay as compared to air drying. Although these modifications are applicable to the RNase protection assay in general, we present an example that used a multiprobe set we developed and have used effectively in the analysis of cytokine mRNA regulation in the brain.