A New Method to Isolate Total dsRNA

  • Go Atsumi
  • Ken-Taro Sekine
  • Kappei Kobayashi
Part of the Methods in Molecular Biology book series (MIMB, volume 1236)


When a diseased plant is suspected to be infected with unknown viruses, the approach of isolating double-stranded RNA (dsRNA) from diseased tissues and analyzing the sequence has been useful for detecting the viruses. This procedure owes its success to the majority of plant pathogenic viruses being RNA viruses, which accumulate dsRNAs as copies of their genome or as a replicative intermediate in infected cells. Conventional dsRNA isolation methods (e.g., chromatography using CF-11 cellulose) require a significant amount of plant material and are laborious and time consuming. Therefore, it has been impractical to isolate dsRNA from many samples at the same time. To overcome these problems, we developed a novel dsRNA isolation method involving a recombinant dsRNA-binding protein. Using this method, we can readily isolate viral dsRNA from a small amount of plant material, and can process numerous samples simultaneously. Purified dsRNA can be used as a template for cDNA synthesis and sequencing, enabling detection of both known and unknown viruses.

Key words

Diagnosis RNA virus Replicative intermediate dsRNA isolation dsRNA-binding protein 



We thank Reiko Tomita for technical advice.


  1. 1.
    Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491PubMedCrossRefGoogle Scholar
  2. 2.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Clark MF, Adams AN (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483PubMedCrossRefGoogle Scholar
  4. 4.
    Fauquet CM, Mayo M, Maniloff J, Desselberger U, Ball LA (2005) Virus taxonomy: VIIIth report of the International Committee on Taxonomy of Viruses. Online via Elsevier, AccessGoogle Scholar
  5. 5.
    Dodds JA, Morris TJ, Jordan RL (1984) Plant viral double-stranded RNA. Annu Rev Phytopathol 22:151–168CrossRefGoogle Scholar
  6. 6.
    Morris TJ, Dodds JA, Hillman B, Jordan RL, Lommel SA, Tamaki SJ (1983) Viral specific dsRNA: diagnostic value for plant virus disease identification. Plant Mol Biol Rep 1:27–30CrossRefGoogle Scholar
  7. 7.
    Valverde R, Nameth S, Jordan RL (1990) Analysis of double-stranded RNA for plant virus diagnosis. Plant Dis 74:255–258CrossRefGoogle Scholar
  8. 8.
    Kobayashi K, Tomita R, Sakamoto M (2009) Recombinant plant dsRNA-binding protein as an effective tool for the isolation of viral replicative form dsRNA and universal detection of RNA viruses. J Gen Plant Pathol 75:87–91CrossRefGoogle Scholar
  9. 9.
    Fedoroff NV (2002) RNA-binding proteins in plants: the tip of an iceberg? Curr Opin Plant Biol 5:452–459PubMedCrossRefGoogle Scholar
  10. 10.
    Eamens AL, Curtin SJ, Waterhouse PM (2011) The Arabidopsis thaliana double-stranded RNA binding (DRB) domain protein family. In: Erdmann VA and Barciszewski J (eds) Non coding RNAs in plants. Springer-Verlag, Berlin, Heidelberg, pp 385–406Google Scholar
  11. 11.
    Hiraguri A, Itoh R, Kondo N, Nomura Y, Aizawa D, Murai Y, Koiwa H, Seki M, Shinozaki K, Fukuhara T (2005) Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol Biol 57:173–188PubMedCrossRefGoogle Scholar
  12. 12.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159PubMedCrossRefGoogle Scholar
  13. 13.
    Kobayashi K, Atsumi G, Iwadate Y, Tomita R, Chiba K, Akasaka S, Nishihara M, Takahashi H, Yamaoka N, Nishiguchi M, Sekine KT (2013) Gentian Kobu-sho-associated virus: a tentative, novel double-stranded RNA virus that is relevant to gentian Kobu-sho syndrome. J Gen Plant Pathol 79:56–63CrossRefGoogle Scholar
  14. 14.
    Atsumi G, Tomita R, Kobayashi K, Sekine KT (2013) Prevalence and genetic diversity of an unusual virus associated with Kobu-sho disease of gentian in Japan. J Gen Virol 94:2360–2365PubMedCrossRefGoogle Scholar
  15. 15.
    Kobayashi K, Atsumi G, Yamaoka N, Sekine KT (2012) Sequencing-based virus hunting and virus detection. Jpn Agric Res Q 46:123–128CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Iwate Biotechnology Research CenterKitakamiJapan
  2. 2.Faculty of AgricultureEhime UniversityMatsuyamaJapan

Personalised recommendations