Nuclear run-on analysis of transcription.

The methods described in Chapters 39, 40, 43, and 44 can only provide information about steady-state levels of transgene RNA. Any differences in specific RNA levels observed in different tissues of a transgenic organism, or any changes in RNA level as a consequence of a physiological or developmental change, cannot, using these techniques, be ascribed to transcriptional controls. Such differences could equally be a consequence of posttranscriptional mechanisms that govern RNA stability. The nuclear run-on assay is, however, a direct, accurate measure of the level of transcription of a particular gene, and can be used to quantitatively measure differences in transgene transcription as a consequence of tissue-specific, developmental or physiological regulation. The method depends on the in vitro incorporation of radioactive ribonucleotides into RNA by RNA polymerase II complexes associated with nascent RNA chains within intact, isolated nuclei. The number of RNA polymerase complexes associated with a particular gene is a measure of the rate of transcription of that gene. Thus, the incorporation of radioactive ribonucleotides into a particular RNA is a measure of the rate of transcription. The level of transcription of a particular gene is assayed by isolating the labeled RNA from nuclei following in vitro incubation with radioactive precursors and using this RNA to probe specific cloned DNAs fixed to a matrix. The level of hybridization is directly related to the level of transcription of the gene of interest, and can be measured by scintillation counting or densitometric scanning following autoradiography. The procedures described here have been developed in this laboratory by adapting previously published protocols.