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Cloning New Small RNA Sequences

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

Abstract

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 

Notes

Acknowledgments

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

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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

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