Nuclear run-on assays: Assessing transcription by measuring density of engaged RNA polymerases

This chapter describes the use of nuclear run-on assays as a direct measure of the density of elongating RNA polymerases. Assuming a relatively constant rate of elongation, this density provides a measure of transcription at the moment of nuclei isolation. Transcriptionally engaged RNA polymerases in isolated nuclei are allowed to continue elongation in the presence of labeled nucleoside triphosphate. The labeled RNA generated from a specific gene sequence is then quantified by hybridization to specific unlabeled DNAs bound to filters. The levels of hybridization to these DNAs, which are present in vast numbers over specific labeled RNAs, are proportional to polymerase density on these sequences in the isolated nuclei. Probing several DNAs simultaneously by simply spotting the single-stranded DNA sequence on a filter and hybridizing the labeled run-on RNA allow the relative density of transcriptionally active polymerase on different genes or on fragments within a single gene to be compared directly. The chapter describes the standard protocols used by researchers in a variety of cell and animal systems. It also describes protocols used with mammalian cells and with yeast for different cell or tissue types.