A New Method to Isolate Total dsRNA

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1236)

Abstract

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 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

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

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