Gel electrophoresis of RNA under denaturing conditions

Abstract

1. We have studied the electrophoretic mobility of RNAs in acrylamide gels under denaturing conditions, using 8 M urea and low salt buffer at 60 °C. In these “urea gels” the mobilities of a number of RNAs in the molecular weight range of 0.5 · 10 6–1.5 · 10 6 are inversely related to their log molecular weights. 2. We have determined molecular weights in “urea gels” of a number of RNAs for which controversial estimates were available, including phage Q β RNA (1.4 · 10 6), cell-sap rRNAs of Tetrahymena pyriformis ( 0.59+0.59 · 10 6 and 0.52 · 10 6) and Crithidia luciliae ( 0.83+0.56 · 10 6 and 0.74 · 10 6) and some mitochondrial rRNAs. 3. We have redetermined the molecular weights of the large subunit rRNAs from Escherichia coli (1.13 · 10 6) and rat-liver cell sap (1.57 · 10 6), phage MS2 RNA (1.23 · 10 6) and the small subunit rRNA from yeast cell sap (0.72 · 10 6) by sedimentation equilibrium. Only the latter value is significantly different from that reported by others (0.62 · 10 6). 4. For a number of viral and rRNAs relative gel electrophoretic mobilities were determined at a range of temperatures, in a standard neutral salt buffer. No simple relationship was found between the variations in mobility and the gross characteristics of the RNAs. Differences in relative gel electrophoretic mobilities of RNAs in the Tris-phosphate-EDTA buffer introduced by Loening, and the Tris-borate-EDTA buffer introduced by Peacock and Dingman, can be attributed to differences in ionic concentrations.