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Utilization of the Citric Acid Procedure and Zonal Ultracentrifugation for Mass Isolation of Nuclear RNA from Walker 256 Carcinosarcoma¹

Department of Pharmacology and the Tumor By-Products Laboratory, Baylor University College of Medicine, Houston, Texas

Nuclear preparations from the Walker tumor were isolated after homogenization of the tumors in 2.5% citric acid. The recovery of nuclei in the procedure employed was 43%, and the purity of the preparations was 85–90% by particle counts with the aid of phase microscopy. The DNA, RNA, and protein contents of the isolated tumor nuclei were 11.9, 5.7, and 34.4 pg per nucleus, respectively. The RNA/DNA ratio of isolated nuclei of the Walker tumor was 0.48, as compared to 0.28 for the nuclei of the normal liver. In studies on normal liver nuclei, it was found that the amounts of DNA, RNA, and protein recovered after treatment with citric acid were 85, 80, and 70%, respectively, of the amounts recovered in the nuclei isolated with the sucrosecalcium procedure. The RNA/DNA ratio was approximately the same. The amount of DNA recovered in the tumor nuclei isolated with the citric acid procedure was 80% of that found when the nuclei were isolated with the sucrose-calcium procedure.

Sucrose density gradient sedimentation studies showed that the nuclei of tumor cells isolated with the citric acid procedure contained approximately of the total nuclear RNA in the 35 S, 45 S, and 55 S fractions. Following a 20-min pulse of orthophosphate-³²P, the radioactivity in the RNA was largely in the high molecular weight fraction. In studies with the zonal ultracentrifuge, fractionation of the RNA was improved, and substantial amounts of tumor nuclear RNA could be separated into various sedimentation classes with this instrument.

¹ These studies were supported in part by grants from the American Cancer Society, the National Science Foundation, the Jane Coffin Childs Fund and the USPHS (CA 08182).

Received 10/20/65.