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Cloning of Short Interfering RNAs from Virus-Infected Plants

  • Thien X. Ho
  • Rachel Rusholme
  • Tamas Dalmay
  • Hui Wang
Part of the Methods in Molecular Biology™ book series (MIMB, volume 451)

Abstract

During their infection in plants, viruses can form double stranded (ds) RNA structures. These dsRNAs can be recognized by plants as “aberrant” signals and short interfering RNA (siRNA) molecules of 19–25 nt will be produced with sequences derived from the viral source. Knowledge about antiviral siRNA profiles including siRNA size, distribution, polarity, etc. provides valuable insights to plant-virus interactions. In this chapter, we describe a simple method for cloning siRNA from virus-infected plants. This protocol includes isolation of small RNAs, their ligation to a pair of 5′ and 3′ adapters, RT-PCR/PCR amplification, and subsequent concatamerization before pGEM-T cloning and sequencing. Concatamers containing as many as 15 small RNA inserts can be produced. This protocol has successfully been apphed to leaf materials of monocots and dicots infected with poty-, carmo-, and sobemo-viruses.

Keywords

RNA silencing siRNA cloning siRNA profile Plant—virus interaction 

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Copyright information

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

Authors and Affiliations

  • Thien X. Ho
    • 1
  • Rachel Rusholme
    • 2
  • Tamas Dalmay
    • 2
  • Hui Wang
    • 3
  1. 1.Centre for Ecology and Hydrology (CEH) OxfordNERCOxford OX1 3SRUK
  2. 2.School of Biological SciencesUniversity of East AngliaNorwich NR4 7TJUK
  3. 3.Department of BiochemistryUniversity of OxfordOxford OX1 3QUUK

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