Run-off transcription
A run-off transcription assay is an assay in molecular biology which is conducted "in vitro" to identify the position of the transcription start site (1 base pair upstream) of a specific promoter along with its accuracy and rate of "in vitro" transcription. Run-off transcription can be used to quantitatively measure the effect of changing promoter regions on in vitro transcription levels, Because of its in vitro nature, however, this assay cannot accurately predict cell-specific gene transcription rates, unlike in vivo assays such as nuclear run-on. To perform a run-off transcription assay , a gene of interest, including the promoter, is cloned into a plasmid. The plasmid is digested at a known restriction enzyme cut site downstream from the transcription start site such that the expected mRNA run-off product would be easily separated by gel electrophoresis. DNA needs to be highly purified prior to running this assay. Additionally, it can be used to detect whether or not transcription is carried out under certain conditions (i.e. in the presence of different chemicals).