The nuclear run-on assay was developed as a method for establishing that the transcription initiation rate contributes to the regulated expression of mammalian genes. The difference between monitoring gene expression by the nuclear run-on assay versus most other assays is that the nuclear run-on assay provides a measure of the frequency of transcription initiation and is largely independent of the effects of RNA stability. It can also be used to determine whether polymerase pausing or attenuation contributes to gene regulation. Briefly, the nuclear run-on assay begins with samples of cells that contain different steady-state amounts of the mRNA or protein of interest. The cells are chilled, and the plasmid membranes are permeabilized or lysed. These steps result in polymerase pausing. The nuclei are then incubated for a short time at 37 C in the presence of nucleoside triphosphates (NTPs) and radiolabeled uridine 5′-triphosphate (UTP). New transcripts are not initiated during this incubation, but the radiolabeled nucleotide becomes incorporated into transcripts that were being synthesized when the cells were first chilled and lysed. The number of nascent transcripts on the gene at the time of chilling is thought to be proportional to the frequency of transcription initiation. To determine the relative number of nascent transcripts in each sample, the radiolabeled RNA is purified and hybridized to a membrane containing immobilized DNA from the gene of interest. The amount of radioactivity that hybridizes to the membrane is approximately proportional to the number of nascent transcripts.