Northern Blotting Techniques for Small RNAs

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1456)

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 specificity 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.

Key words

RNA silencing Northern blot RNA hybridization Small RNA siRNA miRNA 
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Notes

Acknowledgements

Many thanks to Frederick Meins, Jr. and Craig Pikaard for providing support and facilities for experiments shown in this chapter. Azeddine Si-Ammour and Hanspeter Schöb refined the northern blot techniques described here. Mikhail Pooggin and Thomas Hohn provided materials for the viral experiments. Mike Dyer cared for leafy plants, Pierre-François Perroud provided moss tissue, and Kathryn Huisinga supplied Drosophila embryos. Tatiana Simon and Luciano Marpegan provided mouse liver. Franck Vazquez, Mikhail Pooggin, and Andrzej Wierzbicki provided critical comments on the first edition of this book chapter. 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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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Institut de Biologie Moléculaire des PlantesCentre National de la Recherche Scientifique (CNRS) UPR2357Strasbourg CedexUSA

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