Cloning New Small RNA Sequences

  • Yuko Tagami
  • Naoko Inaba
  • Yuichiro WatanabeEmail author
Part of the Methods in Molecular Biology™ book series (MIMB, volume 631)


Small RNAs are key molecules in RNA silencing pathways that exert sequence-specific regulation of gene expression and chromatin modifications in many eukaryotes. In plants, endogenous small RNAs, including microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) play an important role in biological processes such as development and stress responses. In addition, viral genome-derived siRNAs are produced during viral infection, and they exhibit anti-viral defense by an RNA silencing pathway. These endogenous and exogenous small RNAs are mainly 21-24 nucleotides in length. Here, we describe a method to identify small RNA sequences from plant tissues. Small RNAs are purified by column fractionation and gel excision from total RNAs. These small RNAs are ligated at both termini to DNA/RNA chimeric adapters and reverse-transcribed to produce cDNAs. By the following PCR amplification, BanI restriction sites are added to cDNAs, which enables directional concatamerization. Concatamerized-fragments are cloned and sequenced. This method could be applied to identify small RNA sequences from many sources, e.g., mutant plants, plants in various stress environments, and virus-infected plants.

Key words

Cloning Small RNA siRNA miRNA Virus-derived siRNA Sequencing 



We thank Toshiaki Watanabe (National Institute of Genetics) for helpful advice on the detailed cloning methods.


  1. 1.
    Ramachandran V, Chen X (2008) Small RNA metabolism in Arabidopsis. Trends Plant Sci 13:368–374CrossRefPubMedGoogle Scholar
  2. 2.
    Willmann MR, Poethig RS (2007) Conser­vation and evolution of miRNA regulatory programs in plant development. Curr Opin Plant Biol 10:503–511CrossRefPubMedGoogle Scholar
  3. 3.
    Mlotshwaa S, Prussa GJ, Vance V (2008) Small RNAs in viral infection and host defense. Trends Plant Sci 13:375–382CrossRefGoogle Scholar
  4. 4.
    Okamura K, Chung WJ, Ruby JG, Guo H, Bartel DP, Lai EC (2008) The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453:803–806CrossRefPubMedGoogle Scholar
  5. 5.
    Ghildiyal M, Seitz H, Horwich MD, Li C, Du T, Lee S et al (2008) Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320:1077–1081CrossRefPubMedGoogle Scholar
  6. 6.
    Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, Obata Y et al (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453:539–543CrossRefPubMedGoogle Scholar
  7. 7.
    Tam OH, Aravin AA, Stein P, Girard A, Murchison EP, Cheloufi S et al (2008) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453:534–538CrossRefPubMedGoogle Scholar
  8. 8.
    Kurihara Y, Inaba N, Kutsuna N, Takeda A, Tagami Y, Watanabe Y (2007) Binding of tobamovirus replication protein with small RNA duplexes. J Gen Virol 88:2347–2352CrossRefPubMedGoogle Scholar
  9. 9.
    Tagami Y, Inaba N, Kutsuna N, Kurihara Y, Watanabe Y (2007) Specific enrichment of miRNAs in Arabidopsis thaliana infected with Tobacco mosaic virus. DNA Res 14:227–233CrossRefPubMedGoogle Scholar
  10. 10.
    Pfeffer S, Lagos-Quintana M, Tuschl T (2005) Cloning of small RNA molecules. Curr Protoc Mol Biol Chapter 26, Unit 264Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Life SciencesGraduate School of Arts and Sciences, The University of TokyoTokyoJapan

Personalised recommendations