Luciferase assay.
When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. A commonly used reporter gene is the luciferase gene from the firefly Photinus pyralis. This gene encodes a 61-kDa enzyme that oxidizes D-luciferin in the presence of ATP, oxygen, and Mg(++), yielding a fluorescent product that can be quantified by measuring the released light. Including coenzyme A in the reaction enhances the sensitivity of the assay and provides a sustained light reaction. In this protocol, cells transfected with a luciferase reporter plasmid are lysed using a detergent-containing buffer. Cell debris is removed by microcentrifugation and luciferase activity is measured using a luminometer. Some luminometers directly inject the reagents into the cell lysate. Such automation allows the signal to be measured at a precise time following injection, which can increase the consistency of the results. For manual luminometers, the substrate solution is mixed by hand with the cell lysate, and the fluorescence is read at a defined time following mixing. The luciferase assay is extremely rapid, simple, relatively inexpensive, sensitive, and possesses a broad linear range.
Smale
Stephen T
ST
D000818:Animals; D019677:Artificial Gene Fusion; D002457:Cell Extracts / Q000302:isolation & purification; D003584:Cytological Techniques / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D049409:Luciferases, Firefly / Q000032:analysis*; D008163:Luminescent Measurements / Q000379:methods*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D013194:Staining and Labeling / Q000379:methods*; D014158:Transcription, Genetic
D002457:Cell Extracts; C485197:luciferase, Photinus; D049409:Luciferases, Firefly