Advertisement

Detecting sRNAs by Northern Blotting

  • Sara López-GomollónEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 732)

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.

Key words

Short RNAs MicroRNAs Gene expression regulation Gene silencing RNAi RNA silencing Northern blot Transference Hybridisation Nucleic acid detection 

Notes

Acknowledgments

The author wishes to thank Dr. Alvarez for reading the manuscript. S.L-G is supported by a Spanish Government postdoctoral fellowship.

References

  1. 1.
    Phillips, J. R., Dalmay, T., and Bartels, D. (2007) The role of small RNAs in abiotic stress, FEBS Lett 581, 3592–3597.PubMedCrossRefGoogle Scholar
  2. 2.
    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.PubMedCrossRefGoogle Scholar
  3. 3.
    Tomari, Y., and Zamore, P. D. (2005) Perspective: machines for RNAi, Genes Dev 19, 517–529.PubMedCrossRefGoogle Scholar
  4. 4.
    Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., and Bartel, D. P. (2002) MicroRNAs in plants, Genes Dev 16, 1616–1626.PubMedCrossRefGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    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.PubMedCrossRefGoogle Scholar
  7. 7.
    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.PubMedCrossRefGoogle Scholar
  8. 8.
    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.PubMedCrossRefGoogle Scholar
  9. 9.
    Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal Biochem 162, 156–159.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis, J Mol Biol 98, 503–517.PubMedCrossRefGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    Vester, B., and Wengel, J. (2004) LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA, Biochemistry 43, 13233–13241.PubMedCrossRefGoogle Scholar
  14. 14.
    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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  1. 1.School of Biological SciencesUniversity of East AngliaNorwichUK

Personalised recommendations