Gene expression profiling of changes in total RNA levels has provided invaluable knowledge on the regulation of gene expression. Studies on the kinetics of this regulation, however, have been limited by the fact that total cellular RNA is a poor template for revealing short-term changes in gene expression, alterations in RNA decay rates and the kinetics of RNA processing as well as the differentiation thereof. Here, we describe the metabolic labeling and purification of newly transcribed RNA with 4-thiouridine to study the molecular mechanisms governing RNA synthesis, processing, and decay.