Pgl4.23 luc2 pmin luciferase reporter construct

To examine the effects of recurrent somatic mutations on transcription, wild-type and mutant regions 201bp in length and centered on each mutation were synthesized and cloned into the KpnI and NheI site of the pGL4.23[luc2/pmin] luciferase reporter construct (Promega). Lung adenocarcinoma (NCI-H1437), esophageal adenocarcinoma (KYSE-450) and bladder carcinoma (Ku-19-19) cells, growing in 96-well plates, were transfected in quadruplicate with 200ng of the pGL4.23 reporter construct and 40ng of the pRL-CMV Renilla control plasmid (Promega). Forty-eight hours post-transfection luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega). Statistically significant differences in the relative luciferase activities between wild-type and mutant regions were determined using a two-sided Student’s t-Test, assuming equal variance. Visually the variance appears equal between the tested data and there is no biologically reason they should be different.