Preparation of messenger RNA from mature skeletal and cardiac muscle.

Abstract

Messenger RNA (poly(A) + RNA) has been prepared from mature skeletal muscle of adult rats by several procedures. These procedures involved oligodT-cellulose chromatography of either muscle polyribosomes (method 1), polysomal RNA isolated by CsCl gradient centrifugation of muscle polysomes (method 2), total muscle RNA isolated by CsCl gradient centrifugation (method 3), or total muscle RNA prepared by phenol extraction (method 4). Each procedure produced RNA in significant yields which sedimented in a broad band (from 4S to greater than 28S) on sodium dodecyl sulfate--sucrose gradients. In addition, poly (A) + RNA from each procedure stimulated protein synthesis in the wheat germ cell-free system. The best combination of yield and messenger activity was obtained for poly (A) + RNA prepared from polysomal RNA by method 2. This poly(A) + RNA preparation stimulated the cell-free synthesis of a number of presumptive myofibrillar proteins, including myosin heavy chain and actin, in the wheat germ system. The presence of the latter protein among the cell-free products was confirmed by DNase I affinity chromatography of appropriate reaction mixtures. Poly(A) + RNA was also isolated from rat cardiac muscle polysomal RNA by method 2. This RNA also directed the synthesis of myofibrillar proteins in the wheat germ system. The relative amounts of the proteins synthesized in the presence of skeletal and cardiac muscle poly(A) + RNA have been compared. The data indicate that the method described is suitable for the isolation of RNA with message activity from mature mammalian muscle.