Northern Blotting Techniques for Small RNAs
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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.
Key wordsRNA silencing Northern blot RNA hybridization Small RNA siRNA miRNA
Many thanks to Azeddine Si-Ammour and Hanspeter Schöb for refining techniques described here, and to Frederick Meins, Jr., and Craig Pikaard for providing support and facilities for experiments shown in this chapter. Thanks to Mikhail Pooggin, Thomas Hohn, and Dominique Robertson for generating materials and ideas behind the viral experiments. Franck Vazquez, Mikhail Pooggin, and Andrzej Wierzbicki provided critical comments on the manuscript. Mike Dyer cared for leafy plants, while Pierre-François Perroud provided moss tissue. Kathryn Huisinga supplied Drosophila embryos. Tatiana Simon and Luciano Marpegan provided mouse liver. This work was supported by a Friedrich Miescher Institute student fellowship, and postdoctoral fellowships from the Swiss National Foundation and Novartis Foundation.
- 1.Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- 6.Brown T, Mackey K, Du T (2004) Analysis of RNA by northern and slot blot hybridization. Curr Protoc Mol Biol Chapter 4: Unit 4 9
- 31.Meins F Jr (1996) Epigenetic modifications and gene silencing in plants. In: Russo V, Martienssen R, Riggs A (eds) Epigenetic mechanisms of gene regulation. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp 415–442Google Scholar