Northern blotting techniques for small RNAs

Abstract

Cells have evolved intricate RNA-directed mechanisms that destroy viruses, silence transposons, and regulate gene expression. These nucleic acid surveillance and gene silencing mechanisms rely upon the selective base-pairing of ~19-25 nt small RNAs to complementary RNA targets. This chapter describes northern blot hybridization techniques for the detection of such small RNAs. Blots spiked with synthetic standards are used to illustrate the detection specifi city and sensitivity of DNA oligonucleotide probes. Known endogenous small RNAs are then analyzed in samples prepared from several model plants, including Arabidopsis thaliana, Nicotiana benthamiana, Oryza sativa, Zea mays, and Physcomitrella patens, as well as from the animals Drosophila melanogaster and Mus musculus. Finally, the value of northern blotting for dissecting small RNA biogenesis is shown using an example of virus infection in A. thaliana.