Antiviral RNAi

Matthias John, Sébastien Pfeffer

Mammalian host cells and their viral pathogens express and make use of short noncoding RNA molecules to control the infectious cycle. In order to understand their physiological role, it is necessary to develop tools for detection and quantification of these molecules. Here, we present a simple, specific, and very sensitive protocol using short radioactive DNA oligonucleotides for hybridization to homologous RNA target in a nuclease protection assay. The S1 nuclease from Aspergillus oryzae degrades single-stranded oligonucleotides composed of either deoxynucleotides or ribonucleotides. In contrast, double-stranded DNA, double-stranded RNA, or DNA-RNA hybrids are resistant to digestion. Subsequent analysis of the protected DNA oligonucleotide with denaturing gel electrophoresis results in radioactive signals strictly proportional to the abundance of short RNA in a given sample. The protocol works equally well for in vitro cell culture assays and for tissue samples obtained from in vivo experiments.