EXTRACTION OF DOUBLE-STRANDED-RNA FROM PLANT-TISSUES WITHOUT THE USE OF ORGANIC-SOLVENTS

Double-stranded RNAs (dsRNAs) are commonly isolated from plants and insects with toxic organic solvents such as phenol and chloroform that denature and remove proteins from aqueous solutions containing nucleic acids. This procedure was modified by replacing the toxic solvents with sodium dodecyl sulfate and potassium acetate (SDS/KOAc). Samples were homogenized in liquid nitrogen mixed with buffer and SDS at 65 C and then with KOAc at 0 C. A precipitate, consisting of SDS, KOAc, and proteins, was removed by centrifugation. The supernatant containing nucleic acids was collected and subjected to standard CF-11 cellulose chromatography to separate dsRNAs from other nucleic acids. The SDS/KOAc method produced comparable yields and purity of dsRNA products compared to the phenol/chloroform method and is considered a convenient, effective alternative for isolating dsRNA from plant tissues.