Advertisement

RNAi pp 173-185 | Cite as

Identification and Expression Analysis of Small RNAs During Development

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 442)

Summary

RNA interference (RNAi) is a sequence-specific gene regulatory mechanism in which the specificity is determined by small RNAs. Three major classes of endogenous small RNAs, namely microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs/gsRNAs), have been characterized in vertebrates. The miRNAs are mainly involved in development and differentiation and alter gene expression through translational repression or mRNA cleavage. The siRNAs, in contrast, mainly defend against molecular parasites including viruses, transposons, and transgenes. We reported on the expression profile of miRNAs during Xenopus development using a combination of cloning and Northern blot analysis of stage-specific small RNAs. The expression of most miRNAs appeared to be regulated, and some were only expressed at specific stages of development. We also reported on small RNAs specifically expressed during gametogenesis in the mouse. The study revealed the existence of retrotransposon-derived siRNAs in oocytes and a novel class of small RNA (piRNAs/gsRNAs) in testes. In this chapter, we describe methods of low molecular weight RNA preparation, small RNA cloning, annotation of small RNAs, and analysis of expression during development.

Key Words

miRNA siRNA rasiRNA piRNA RNAi development small RNA 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

The authors would like to thank T. Okuno, K. Mise, A. Takeda, Y. Watanabe, and Y. Kurihara for technical assistance.

References

  1. 1.
    Bartel, D. P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116, 281–297.CrossRefPubMedGoogle Scholar
  2. 2.
    Sijen, T., and Plasterk, R. H. (2003). Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi. Nature 426, 310–314.CrossRefPubMedGoogle Scholar
  3. 3.
    Reinhart, B. J., Slack, F. J., Basson, M., et al. (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906.CrossRefPubMedGoogle Scholar
  4. 4.
    Ambros, V. (2003). MicroRNA pathways in flies and worms: Growth, death, fat, stress, and timing. Cell 113, 673–676.CrossRefPubMedGoogle Scholar
  5. 5.
    Yekta, S., Shih, I. H., and Bartel, D. P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596.CrossRefPubMedGoogle Scholar
  6. 6.
    Giraldez, A. J., Mishima, Y., Rihel, J., et al. (2006). Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75–79.CrossRefPubMedGoogle Scholar
  7. 7.
    Giraldez, A. J., Cinalli, R. M., Glasner, M. E., et al. (2005). MicroRNAs regulate brain morphogenesis in zebrafish. Science 308, 833–838.CrossRefPubMedGoogle Scholar
  8. 8.
    Bernstein, E., Kim, S. Y., Carmell, M. A., et al. (2003). Dicer is essential for mouse development. Nat. Genet. 35, 215–217.CrossRefPubMedGoogle Scholar
  9. 9.
    Watanabe, T., Takeda, A., Mise, K., et al. (2005). Stage-specific expression of microRNAs during Xenopus development. FEBS Lett. 579, 318–324.CrossRefPubMedGoogle Scholar
  10. 10.
    Watanabe, T., Takeda, A., Tsukiyama, T., et al. (2006). Identification and characterization of two novel classes of small RNAs in the mouse germline: Retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes. Genes Dev. 20, 1732–1743.CrossRefPubMedGoogle Scholar
  11. 11.
    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.CrossRefPubMedGoogle Scholar
  12. 12.
    Mansfield, J. H., Harfe, B. D., Nissen, R., et al. (2004). MicroRNA-responsive “sensor” transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat. Genet. 36, 1079–1083.CrossRefPubMedGoogle Scholar
  13. 13.
    Lai, E. C., Tam, B., and Rubin, G. M. (2005). Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs. Genes Dev. 19, 1067–1080.CrossRefPubMedGoogle Scholar
  14. 14.
    Leaman, D., Chen, P. Y., Fak, J., et al. (2005). Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121, 1097–1108.CrossRefPubMedGoogle Scholar
  15. 15.
    Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200.CrossRefPubMedGoogle Scholar
  16. 16.
    Ambros, V., Bartel, B., Bartel, D. P., et al. (2003). A uniform system for microRNA annotation. RNA 9, 277–279.CrossRefPubMedGoogle Scholar
  17. 17.
    Kurihara, Y., and Watanabe, Y. (2004). Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc. Natl. Acad. Sci. USA 101, 12753–12758.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  1. 1.Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organizatio of Information and Systems, and Department of GeneticsSchool of Life Science, The Graduate University for Advanced Studies (SOKENDAI)MishimaJapan
  2. 2.Laboratory of Reproductive Biology, Department of AgricultureKyoto UniversityKyotoJapan

Personalised recommendations

Cite protocol

Buy options