Dualluciferase

Dualluciferase, or dual-luciferase assay, is a molecular biology technique used to measure the activity of two luciferase enzymes within a single sample. It is commonly employed to assess promoter or transcriptional element activity while providing an internal control to normalize for sample-to-sample variability such as transfection efficiency or cell viability.

In a typical workflow, cells are co-transfected with two reporter constructs: one encoding firefly luciferase to report the experimental condition, and a second encoding another luciferase (most often Renilla luciferase) as a normalization reference. The assay uses two sequential substrate reactions. First, a firefly substrate (D-luciferin) is added and luminescence is measured. A stop or quenching step then inactivates the firefly signal, after which a second substrate (coelenterazine for Renilla, or alternative substrates for other enzymes) is added to measure the second luminescent signal. Many commercial kits are designed to perform these steps in a single wells with a luminometer capable of rapid sequential readings.

Firefly and Renilla luciferases are the most common pair, due to their distinct substrates and non-overlapping emission spectra. Other luciferases such as Gaussia or NanoLuc can be used in alternative dual-reporter configurations, depending on experimental needs.

Applications include reporter gene assays for gene expression studies, high-throughput screening, and functional analysis of regulatory elements. Advantages include internal normalization, wide dynamic range, and reduced experimental variability. Limitations involve the need for compatible reagents and instrumentation, potential cross-talk between reporters if timing is not precise, and higher cost relative to single-reporter assays.