JEAN LUST
alone. These experiments establish precise conditions for use of SDS and handling of tissue required to obtain a sizable amount of insoluble RNA. Characterization of this RNA strongly suggests that it contains nuclear RNA as well as cytoplasmic RNA.
Methods
Preparation and Disruption of Frozen Powder of Rat Liver by SDS.
SDS was prepared by purification of Duponol C" according to the procedure described by Crestfield, Smith, and Allen (1) and dissolved in 0.0125 M Na,HPO,-0.0125 M NaH,PO, in concentrations varying between 0.1 and 2 gm% (w/v).
Rats from the Long Evans, Nelson ( 27) or Fisher Agouti strains were fasted for 20-24 hr. The livers were removed under ether anesthesia and frozen in a Dewar flask containing dry ice and alcohol. A frozen powder of 0.5 gm of liver Gssue was prepared by crushing frozen liver with a mortar and pestle in a dry-ice chest. A test tube with the powder was introduced quickly into a boiling water bath. Immediately thereafter, 4 ml of SDS, which had reached the temperature of the water bath, were poured on the powder. Heating time with vigorous stirring in the water bath lasted between 3 and 10 min. The stirring device consisted of a narrow shielded electrical wire propelled by a light-duty electric stirrer3 revolving at maximal speed. The SDS-treated tissue was then cooled rapidly in a Dewar flask containing alcohol and dry ice.
Preparation and Disruption of a Rat Liver Homogenate by SDS, In the cold room, thawed liver tissue was ground in a Potter homogenizer fitted with a Teflon pestle after addition of a small volume of solvent consisting of 0.0125M Na,HPO,-0.0125M NaH2P0, buffer solution containing, in some experiments, 0.25M sucrose. In other experiments, the solvent was Hank's solution (11). These solutions were precooled in the cold room. Either 3 ml of the homogenate was heated in a boiling water bath before the addition of SDS heated to 100" or 1 ml of homogenate was added promptly to 3 ml of heated SDS. The subsequent steps were carried out as described above. Preparation of a Suspension and Disruption of HeLa Cells. HeLa cells 'E. I. du Pont de Nemours & Co. 3Eastern stirrer, model 1.