Microgranular Cellulose Improves dsRNA Recovery from Plant Nucleic Acid Extracts

Double-stranded (ds)RNA is an important nonspecific indicator of the presence of RNA viruses in bacteria, fungi and plants (1,3). The dsRNA usually represents either the replicative form or the genome of an RNA virus, or it may be an intermediate in the replication of viroids or satellite RNAs. Its size(s) can provide specific information on the likely identity of a virus-like agent infecting a host, and successful isolation and purification of the dsRNA allows it to be cloned for use as a specific diagnostic molecular probe without the need for purifying the virus (4). Sequence analysis of clones provides information on the likely taxonomic position of a virus-like agent associated with the dsRNA (5) and can lead to identification of the virus. The preferred method for dsRNA isolation uses differential absorption of the dsRNA fraction from nucleic acid extracts to chemically unmodified fibrous cellulose powder (CF11; Whatman, Maidstone, Kent, England, UK) in the presence of sodium chloride and at a specific ethanol concentration. This is followed by washing, elution in ethanol-free buffer, concentration and electrophoresis (1). During an investigation of a sugarcane disease of unknown etiology (sugarcane striate mosaic disease [ScSMD]), in which a disease-associated dsRNA band of about 9 kbp was identified by the routine CF11 procedure, we found that the band intensity in analytical agarose and polyacrylamide gel electropherograms was low and variable. For further study, large amounts of infected leaf were required, and dsRNA had to be isolated from large volumes of nucleic acid extract. We report an improved method for dsRNA isolation that routinely increased yields in the sugarcane system and suggest that it may be applicable to other systems in which the CF11 procedure is unsatisfactory. Microgranular cellulose has been used previously to recover dsRNA from dissolved polyacrylamide gels (2). We therefore tested a more finely divided form of cellulose as a replacement for CF11 cellulose. We used a thin-layer chromatography grade of microgranular cellulose (MN 300 cellulose powder; Macherey-Nagel GmbH, Düren, Germany). Direct substitution of the MN 300 cellulose powder for CF11 cellulose in a modification of the column purification method of Dodds (1) was not successful because of extremely low flow rates through the chromatography column. However, when a batch method was used (Table 1), the yield of 9-kbp dsRNA was consistently at least 10-fold higher than for the CF11 column method (Figure 1). The MN 300 powder also recovered dsRNA from Nicotiana tabacum infected with tobacco mosaic virus and N. glutinosa infected with either alfalfa mosaic virus (Figure 1, lane 4) or cucumber mosaic virus at a higher yield than the CF11 column method. A DNase digestion step was included routinely in this procedure to remove host DNA that tended to co-migrate with the 9-kbp dsRNA. Single-stranded RNA contaminants were obtained with both cellulose materials, and these interfered with the detection of dsRNA smaller than 4 kbp (Figure 1, lanes 2 and 3). These could be removed either by predigestion of the sample with ribonuclease A (Sigma Chemical, St. Louis, MO, USA) (0.5 μg/mL in 0.3 M NaCl, 0.1 M Tris-HCl, pH 7.0 at 37°C for 0.5–1 h) or by an absorption-elution cycle on a CF11 cellulose column (1). RNase A treatment allowed minor subgenomic dsRNAs of 6, 2.6 and 2.5 kbp (which are not visible in Figure 1) to be seen only in the MN 300 cellulose-prepared samples when they were fractionated on agarose gels