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.[1] [2][3]

Run-off transcription can be used to quantitatively measure the effect of changing promoter regions on in vitro transcription levels,[1][2][4] 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.[1][2]

To perform a run-off transcription assay, a gene of interest, including the promoter, is cloned into a plasmid.[4] 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.[1][2][4] DNA needs to be highly purified prior to running this assay.[1] [2] To initiate transcription, radiolabeled UTP, the other nucleotides, and RNA polymerase are added to the linearized DNA.[1][2] Transcription continues until the RNA polymerase reaches the end of the DNA where it simply “runs off” the DNA template, resulting in an mRNA fragment of a defined length.[1][2] This fragment can then be separated by gel electrophoresis, alongside size standards, and autoradiographed.[1][2][4] The corresponding size of the band will represent the size of the mRNA from the restriction enzyme cut site to the transcription start site (+1).[4] The intensity of the band will indicate the amount of mRNA produced.[4]

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).[5]