Isolation and Analysis of Double-Stranded RNA from Virus-Infected Plant and Fungal Tissue

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Abstract
A simple, rapid method for the isolation of double-stranded RNA (dsRNA) from virus-infected plant and fungal tissues provides a new approach to virus detection and identification. Diseased tissue was phenol-extracted to isolate cellular nucleic acids, and viral dsRNA was selectively purified from other nucleic acids by binding to cellulose powder in 15% ethanol either in small columns or by a batch procedure. The product was analyzed first by gel electrophoresis and then by RNase treatment to identify dsRNA. The method permits rapid and efficient isolation and analysis of dsRNA from small amounts (1-10 g) of tissue and from multiple samples using small amounts (0.1-2.5 g) of cellulose powder. Successful isolation of dsRNA does not depend on the type of tissue processed and the method therefore is potentially useful for the study of RNA virus replication and for detection and diagnosis of virus infections directly from the infected host tissues.