Direct determination of the specific activity of RNA uniformly labeled with 32P

A simple method for the direct determination of the specific activity of RNA uniformly labeled with 32P is described. The procedure is based on the premise that upon disintegration of 32P to 32S, the phosphodiester bond is broken. Analysis of the rate of decay of the full-length molecule by gel electrophoresis and autoradiography can accurately determine the "intramolecular specific activity" of the RNA. An equation that predicts the relative intensity of the intact RNA molecules remaining as a function of time is presented. These predictions are confirmed using in vitro-synthesized RNA labeled at a known specific activity. This procedure has been used to determine the intramolecular specific activity of RNA labeled in vivo in yeast. It can also be employed to choose the best conditions for experiments utilizing uniformly labeled RNA or single-stranded DNA and requiring the detection of intact molecules.