Northern Blotting Techniques for Small RNAs

In eukaryotes, RNA silencing encompasses a range of biochemical processes mediated by ∼20–25 nt small RNAs (smRNAs). This chapter describes northern blot hybridization techniques optimized for detection of such smRNAs, whether extracted from plant or animal tissues. The basic protocol is described, and control blots illustrate the detection specificity and sensitivity of this method using DNA oligonucleotide probes. Known endogenous smRNAs are analyzed in samples prepared from several model plant species, including Arabidopsis thaliana , Nicotiana benthamiana , Oryza sativa , Zea mays , and Physcomitrella patens , as well as the animals Drosophila melanogaster and Mus musculus . Finally, the usefulness of northern blotting in dissecting smRNA biogenesis is shown for the particular case of DNA virus infection.