Abstract
Small RNAs (sRNAs) play fundamental roles in modifying the transcriptomes and proteomes of organisms. sRNAs can be classified according to their origin and way of action into classes such as microRNAs, trans-acting siRNAs, heterochromatin siRNAs, Piwi-interacting RNAs, and natural antisense siRNAs, although microRNAs are the most conserved and studied class. The expression pattern of a sRNA can be indicative of its biological function. Northern blotting is one of the most widely used methods to identify, validate, and study the expression profile of sRNAs because it is a quantitative, relatively inexpensive technique that is readily available for most laboratories. This chapter describes a protocol for sRNA Northern blot analysis, which includes RNA extraction, polyacrylamide gel electrophoresis, hybridisation, or the detection of sRNA using oligonucleotide probes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Phillips, J. R., Dalmay, T., and Bartels, D. (2007) The role of small RNAs in abiotic stress, FEBS Lett 581, 3592–3597.
Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S., and Kim, V. N. (2003) The nuclear RNase III Drosha initiates microRNA processing, Nature 425, 415–419.
Tomari, Y., and Zamore, P. D. (2005) Perspective: machines for RNAi, Genes Dev 19, 517–529.
Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., and Bartel, D. P. (2002) MicroRNAs in plants, Genes Dev 16, 1616–1626.
Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L., and Poethig, R. S. (2004) SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis, Genes Dev 18, 2368–2379.
Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory, A. C., Hilbert, J. L., Bartel, D. P., and Crete, P. (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs, Mol Cell 16, 69–79.
Lu, C., Kulkarni, K., Souret, F. F., MuthuValliappan, R., Tej, S. S., Poethig, R. S., Henderson, I. R., Jacobsen, S. E., Wang, W., Green, P. J., and Meyers, B. C. (2006) MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant, Genome Res 16, 1276–1288.
Borsani, O., Zhu, J., Verslues, P. E., Sunkar, R., and Zhu, J. K. (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis, Cell 123, 1279–1291.
Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal Biochem 162, 156–159.
Kumar, G. N., Iyer, S., and Knowles, N. R. (2007) Extraction of RNA from fresh, frozen, and lyophilized tuber and root tissues, J Agric Food Chem 55, 1674–1678.
Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis, J Mol Biol 98, 503–517.
Pall, G. S., Codony-Servat, C., Byrne, J., Ritchie, L., and Hamilton, A. (2007) Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by Northern blot, Nucleic Acids Res 35, e60.
Vester, B., and Wengel, J. (2004) LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA, Biochemistry 43, 13233–13241.
Valoczi, A., Hornyik, C., Varga, N., Burgyan, J., Kauppinen, S., and Havelda, Z. (2004) Sensitive and specific detection of microRNAs by Northern blot analysis using LNA-modified oligonucleotide probes, Nucleic Acids Res 32, e175.
Acknowledgments
The author wishes to thank Dr. Alvarez for reading the manuscript. S.L-G is supported by a Spanish Government postdoctoral fellowship.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Humana Press
About this protocol
Cite this protocol
López-Gomollón, S. (2011). Detecting sRNAs by Northern Blotting. In: Dalmay, T. (eds) MicroRNAs in Development. Methods in Molecular Biology, vol 732. Humana Press. https://doi.org/10.1007/978-1-61779-083-6_3
Download citation
DOI: https://doi.org/10.1007/978-1-61779-083-6_3
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-082-9
Online ISBN: 978-1-61779-083-6
eBook Packages: Springer Protocols