Use of CH3HgOH-agarose gels for the electrophoresis of heterogeneous nuclear RNA and messenger RNA from mammalian cells

Abstract The size distribution of labeled hnRNA and mRNA from Hela cells and mouse Ehrlich ascites cells has been analyzed by gel electrophoresis in the presence of the denaturing agent CH 3 HgOH. Up to 43% of the mass of total hnRNA consisted of molecules at least 28 kilobases in size, provided this RNA had been extracted under conditions minimizing degradation. Ten to sixteen percent of the mass of polyadenylated hnRNA consisted of molecules larger than 13 kilobases, although the average molecular weight was less than that of total hnRNA. In comparison, more than 90% of the mass of labeled mRNA (total or polyadenylated) consisted of molecules smaller than 5.5 kilobases. A linear relationship was observed between appropriate functions of molecular weight and mobility for RNA molecules up to at least 13 kilobases in size, provided a low gel concentration and voltage gradient were employed. Increasing these parameters resulted in anomalously fast migration of large RNA molecules. Electrophoretic profiles were not significantly altered when the amount of poly(A) hnRNA was increased from about 1 to 50 μg per cylindrical gel. Thus CH 3 HgOH-agarose gel electrophoresis can be used preparatively for the fractionation of hnRNA. Aggregates containing labeled mRNA (formed in vitro in the presence of excess nuclear RNA) were dissociated by CH 3 HgOH. Partial degradation of high molecular weight RNA was observed after prolonged exposure to CH 3 HgOH.