<P>To the aqueous layer, 1/2 volume of phenol was added; the mixture was shaken in a Thomas Rotator for an additional 1/2 hour, and centrifuged at 10,000 x g for 10 minutes. Two and one half volumes of 95 percent ethanol and 1/10 volume of 1.0M K acetate, pH 5.0, were added to the resulting aqueous layer, and RNA was precipitated overnight in a freezer. This preparation was labeled aqueous RNA and represents cytoplasmic RNA.
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<P>To the interphase and the phenol layers, was added 1/2 volume of H2O that was adjusted to 0.2 per cent with respect to SDS and 0.01 per cent with respect to hydroxyquinoline, para-aminosalicylic acid, and naphthalene disulfate. The mixture was shaken in a Burrell Shaker for 35 minutes and centrifuged at 10,000 x g for 10 minutes. One-half volume of phenol and 1/2 volume of water were added to the aqueous phase and the interphase. The mixture was shaken on a Burrell Shaker for 35 minutes. Again centrifugation was carried out at 10,000 x g for 10 minutes; 2.5 volume of 95 percent ethanol and 1/10 volume of 1.0M K acetate, pH 5, were added to the aqueous phase and RNA was allowed to precipitate overnight. This preparation was labeled interphase RNA and represents primarily nuclear RNA.