In vivo run-on assays to monitor nascent precursor RNA transcripts

Biochemical methods have provided mechanistic insights into the different transcription phases during which the RNA polymerase is assembled at gene promoter and becomes engaged in the elongation of nascent transcripts. Evidence that transcription takes place in specific regions of the nucleus has fuelled the need to develop assays that can be performed in living cells and provide information on the location of the specific foci, where transcription takes place. In this chapter, we describe a method that is based on the incorporation of a fluorine-conjugated uridine analogue, incorporation that can be monitored by immunofluorescence and light microscopy using specific fluorochrome-conjugated monoclonal antibodies. This assay allows direct monitoring of active transcription foci in living cells. When coupled to suitable software, the method outlined here also provides a semiquantitative approach to measure the number of active transcription foci that correlate with the proliferation state of the cell. Therefore, the assay we present here is a sensitive analytical tool to monitor the topology of transcription foci in the eukaryotic cell nucleus and to gain insight into transcription rates.