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Northern Blotting Techniques for Small RNAs

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Plant Epigenetics

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 631))

Abstract

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.

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Acknowledgments

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.

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  1. Pikaard Laboratory, Biology Department, Washington University, Washington, MO, USA

    Todd Blevins

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  1. Todd Blevins
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Editors and Affiliations

  1. Dept. Biological Sciences, University of Lethbridge, University Drive 4401, Lethbridge, T1K 3M4, Canada

    Igor Kovalchuk

  2. Dept. Biological Sciences, University of Lethbridge, University Drive 4401, Lethbridge, T1K 3M4, Canada

    Franz J. Zemp

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Blevins, T. (2010). Northern Blotting Techniques for Small RNAs. In: Kovalchuk, I., Zemp, F. (eds) Plant Epigenetics. Methods in Molecular Biology™, vol 631. Humana Press. https://doi.org/10.1007/978-1-60761-646-7_9

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  • DOI: https://doi.org/10.1007/978-1-60761-646-7_9

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-645-0

  • Online ISBN: 978-1-60761-646-7

  • eBook Packages: Springer Protocols

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