A non-phenol-chloroform extraction of double-stranded RNA from plant and fungal tissues

Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol-chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing P-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method. (C) 2008 Elsevier B.V. All rights reserved.