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ARS Home » Pacific West Area » Corvallis, Oregon » Horticultural Crops Research Unit » Research » Publications at this Location » Publication #222514
Title: A New Method for Extraction of Double-Stranded RNA from Plants

Author
item TZANETAKIS, I - OREGON STATE UNIVERSITY
item Martin, Robert

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/13/2008
Publication Date: 3/15/2008
Citation: Tzanetakis, I.E., Martin, R.R. 2008. A new method for extraction of double-stranded RNA from plants. Journal of Virological Methods. 149:167-170.

Interpretive Summary: High molecular weight double-stranded RNA (dsRNA) in plants is a very strong indication of a virus infection, since with very rare exceptions virus-free plants are free of these molecules. In plants, DNA is copied to make RNA, which is then translated to make proteins. Most plant viruses have RNA genomes rather than DNA and thus make a complimentary RNA during replication. This leads to the production of plus and minus RNA which hybridizes to make dsRNA. The dsRNA is quite stable and readily purified from most plant species. The purified dsRNA is the material of choice for characterizing viruses when the virus itself can not be purified. A number of methods for purifying dsRNA have been published, but for some unknown reason they do not work for purifying dsRNA from blueberry and cranberry. Here we describe a method for purifying dsRNA from blueberry that is very efficient and also works for other plant species. An added advantage of this method is that it does not use phenol, which is a very caustic chemical, or other organic solvents other than ethanol. This method is based on a lithium salt buffer and detergents. The quality of the dsRNA purified is such that it can be used for cloning and sequencing and thus, virus characterization.

Technical Abstract: The presence of high molecular weight double-stranded RNA (dsRNA) in plants is associated with the presence of RNA viruses. DsRNA is stable and can be extracted easily from the majority of plant species and provides an excellent tool for characterization of novel viruses that are recalcitrant to purification. Several protocols have been developed for dsRNA purification, the majority of which are based on extraction with phenol and chloroform. We have developed a protocol for dsRNA extraction based on a lithium salts buffer that does not require organic solvents other than alcohols. The method yields comparable amount of dsRNA to previously described protocols and consistently yields dsRNA from Ericaceous hosts that have been recalcitrant to dsRNA purification using traditional protocols. The quality of the dsRNA purified is such that it can be used for downstream enzymatic reactions including reverse transcription-polymerase chain reaction and cloning.