Single Nucleotide Mapping of RNA 5′ and 3′ Ends

Abstract

Nuclease protection assay is a sensitive method for detection, quantitation, and mapping of a specific RNA in an extremely heterogeneous mixture of RNAs, such as total cellular RNA. The assay is based on a small volume solution hybridization of a single-stranded synthetic antisense and labeled RNA probe to a RNA sample. Thus, it is much more efficient than the common immobilized hybridization on a membrane, such as in northern-blot analysis. After solution hybridization, different nucleases are used to remove any remaining single-stranded nucleotides within the probe and sample RNA by digestion. Then, the remaining probe-target hybrids are purified and separated on a denaturing polyacrylamide gel. Using a radioactive labeled probe, the protected probe can be visualized by direct autoradiography and the copy number can be calculated based on the specific radioactivity of the RNA probe and the length of protected fragment. Because of its high sensitivity and resolution, nuclease protection assay is the most effective procedure for mapping internal and external boundaries in mRNA compared to other RNA detection methods such as RT-PCR.