The Two-Step Purification of Ribosomal RNA and Plant Viral RNA by Polyacrylamide Slab Gel Electrophoresis

Abstract

The requirement for purified plant viral RNAs for sequence characterization by hybridization analysis using complementary DNA led to the development of a routine two-step procedure for their purification. The method was worked out using Escherichia coli ribosomal RNAs and then applied to four plant viral RNAs, all of which contain four major RNA components. The first of the two steps involves the electrophoresis of native RNA on 2·8 % polyacrylamide slab gels, the location of the RNA bands by brief staining and the recovery of the RNA from each gel band by electrophoretic elution. The eluted RNA, concentrated by ethanol precipitation, was then run on a second gel after a denaturation step to release nicked and aggregated RNA fragments. Each RNA band was again located by staining and recovered by electrophoretic elution and ethanol precipitation. The 16-S and 23-S ribosomal RNAs and the plant viral RNAs were obtained in overall yields of 40 and 8-16 % respectively. RNAs so purified have been successfully used for the preparation of complementary DNA by two different methods.