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Small RNA Cloning

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RNA Therapeutics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 629))

Abstract

The next generation sequencing technologies have made the exhaustive sequencing of all small RNA species within a small RNA library possible. Thus, comparative studies on the changes in small-RNA transcriptomes between two biological samples represent a powerful means of revealing the functions of small RNAs in physiological and pathological processes. Here, we describe a small RNA cloning method that can be used to generate small RNA cDNA libraries for both conventional and next generation (deep) sequencing. It can also be used for detection and quantitative analyses of small RNAs using PCR.

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References

  1. Aravin, A.A., Lagos-Quintana, M., Yalcin, A., Zavolan, M., Marks, D., Snyder, B., Gaasterland, T., Meyer, J., and Tuschl, T. (2003) The small RNA profile during Drosophila melanogaster development. Dev Cell, 5, 337–350.

    Article  PubMed  CAS  Google Scholar 

  2. Lee, R.C. and Ambros, V. (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science, 294, 862–864.

    Article  PubMed  CAS  Google Scholar 

  3. Lau, N.C., Lim, L.P., Weinstein, E.G., and Bartel, D.P. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science, 294, 858–862.

    Article  PubMed  CAS  Google Scholar 

  4. Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001) Identification of novel genes coding for small expressed RNAs. Science, 294, 853–858.

    Article  PubMed  CAS  Google Scholar 

  5. Lau, N.C., Seto, A.G., Kim, J., Kuramochi-Miyagawa, S., Nakano, T., Bartel, D.P., and Kingston, R.E. (2006) Characterization of the piRNA complex from rat testes. Science, 313, 363–367.

    Article  PubMed  CAS  Google Scholar 

  6. Grivna, S.T., Beyret, E., Wang, Z., and Lin, H. (2006) A novel class of small RNAs in mouse spermatogenic cells. Genes Dev, 20, 1709–1714.

    Article  PubMed  CAS  Google Scholar 

  7. Girard, A., Sachidanandam, R., Hannon, G.J., and Carmell, M.A. (2006) A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature, 442, 199–202.

    PubMed  Google Scholar 

  8. Aravin, A., Gaidatzis, D., Pfeffer, S., Lagos-Quintana, M., Landgraf, P., Iovino, N., Morris, P., Brownstein, M.J., Kuramochi-Miyagawa, S., Nakano, T., Chien, M., Russo, J.J., Ju, J., Sheridan, R., Sander, C., Zavolan, M., and Tuschl, T. (2006) A novel class of small RNAs bind to MILI protein in mouse testes. Nature, 442, 203–207.

    PubMed  CAS  Google Scholar 

  9. Watanabe, T., Takeda, A., Tsukiyama, T., Mise, K., Okuno, T., Sasaki, H., Minami, N., and Imai, H. (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.

    Article  PubMed  CAS  Google Scholar 

  10. Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V., and Zamore, P.D. (2006) A distinct small RNA pathway silences selfish genetic elements in the germline. Science, 313, 320–324.

    Article  PubMed  CAS  Google Scholar 

  11. Saito, K., Nishida, K.M., Mori, T., Kawamura, Y., Miyoshi, K., Nagami, T., Siomi, H., and Siomi, M.C. (2006) Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev, 20, 2214–2222.

    Article  PubMed  CAS  Google Scholar 

  12. Ro, S., Song, R., Park, C., Zheng, H., Sanders, K.M., and Yan, W. (2007) Cloning and expression profiling of small RNAs expressed in the mouse ovary. RNA, 13, 2366–2380.

    Article  PubMed  CAS  Google Scholar 

  13. Ruby, J.G., Jan, C., Player, C., Axtell, M.J., Lee, W., Nusbaum, C., Ge, H., and Bartel, D.P. (2006) Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell, 127, 1193–1207.

    Article  PubMed  CAS  Google Scholar 

  14. Terns, M.P. and Terns, R.M. (2002) Small nucleolar RNAs: versatile trans-acting molecules of ancient evolutionary origin. Gene Expr, 10, 17–39.

    PubMed  CAS  Google Scholar 

  15. Ouellet, D.L., Perron, M.P., Gobeil, L.A., Plante, P., and Provost, P. (2006) MicroRNAs in gene regulation: when the smallest governs it all. J Biomed Biotechnol, 2006, 69616.

    Article  PubMed  Google Scholar 

  16. Maatouk, D. and Harfe, B. (2006) MicroRNAs in development. ScientificWorldJournal, 6, 1828–1840.

    Article  PubMed  Google Scholar 

  17. Kim, V.N. and Nam, J.W. (2006) Genomics of microRNA. Trends Genet, 22, 165–173.

    Article  PubMed  CAS  Google Scholar 

  18. Kim, V.N. (2006) Small RNAs just got bigger: Piwi-interacting RNAs (piRNAs) in mammalian testes. Genes Dev, 20, 1993–1997.

    Article  PubMed  CAS  Google Scholar 

  19. Guryev, V. and Cuppen, E. (2009 Next-generation sequencing approaches in genetic rodent model systems to study functional effects of human genetic variation. FEBS Lett, 583, 1668–1673.

    Article  PubMed  CAS  Google Scholar 

  20. Ro, S., Park, C., Jin, J., Sanders, K.M., and Yan, W. (2006) A PCR-based method for detection and quantification of small RNAs. Biochem Biophys Res Commun, 351, 756–763.

    Article  PubMed  CAS  Google Scholar 

  21. Ro, S., Park, C., Sanders, K.M., McCarrey, J.R., and Yan, W. (2007) Cloning and expression profiling of testis-expressed microRNAs. Dev Biol, 311, 592–602.

    Article  PubMed  CAS  Google Scholar 

  22. Ro, S., Park, C., Song, R., Nguyen, D., Jin, J., Sanders, K.M., McCarrey, J.R., and Yan, W. (2007) Cloning and expression profiling of testis-expressed piRNA-like RNAs. RNA, 13, 1693–1702.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors would like to thank Jonathan Cho for reading and editing the text. This work was supported by grants from the National Institute of Health (HD048855 and HD050281) to W.Y.

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Ro, S., Yan, W. (2010). Small RNA Cloning. In: Sioud, M. (eds) RNA Therapeutics. Methods in Molecular Biology, vol 629. Humana Press. https://doi.org/10.1007/978-1-60761-657-3_17

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  • DOI: https://doi.org/10.1007/978-1-60761-657-3_17

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-656-6

  • Online ISBN: 978-1-60761-657-3

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