Detecting mRNA by Use of the Ribonuclease Protection Assay (RPA)

The characterization of gene expression patterns within cells and tissues is a very desirable and highly informative way of providing insights into cell development, maintenance, and cell-cell interactions. Several widely used methods, such as Northern or dot blot, in situ hybridization, reverse transcription-polymerase chain reaction (RT- PCR), and ribonuclease protection assay (RPA), have been developed to analyze changing cellular distribution and concentration of mRNAs. The fundamental principle to detect mRNA expression is the specific hybridization of a complementary DNA or RNA probe with its cellular homolog. The relative or absolute quantitation of such mRNAs is complex and each of the techniques has to be judged on its technical limitations and the information it provides. This chapter provides a short introduction to the RPA together with the typical advantages, new nonradioactive approaches, and more simplified gel systems based on original results from our laboratory.