Differentiate separate luciferase signals in a single sample

Luciferase enzymes can be used as single reporters to study one biological event in a given experiment, but because of their different spectral properties and substrates, multiple luciferase enzymes can be combined for multiplex luciferase experiments. With these types of experiments, called dual-reporter assays, two luciferase enzymes are simultaneously expressed and detected in a single sample.

The Thermo Scientific Pierce Dual-Spectral Luciferase Calculator is a simple, downloadable tool for distinguishing luminescent signals from two luciferase reactions measured simultaneously in the same sample. The two luminescent reactions must have distinct emission profiles to allow for at least partial separation of luminescent signals based on wavelength. Luminescence from the two luciferase reactions is measured on any standard filter-based luminometer or multi-modal instrument. Filters should be selected for each luciferase reporter based on their emission spectra to maximize light collected from that reporter while minimizing light from the other reporter. This calculator is ideal for use in cases where stray light from the other reporter cannot be completely filtered out due to incomplete wavelength separation or less-than-ideal filter sets.

Download the Pierce Dual-Spectral Luciferase Calculator

Note: In cases where the light emission from one reporter can be distinctly separated from light emission from the other reporter using filters alone, the calculator is not required and may in fact introduce unnecessary error into the data collected.


Pierce Dual-Spectral Luciferase Calculator algorithms

The Pierce Dual-Spectral Luciferase Calculator is based on the following linear algorithm

which may be written as



Fg Total luminescence from the green filter
Fr Total luminescence from the red filter
KGg Transmission coefficient of green luciferase through green filter
KRg Transmission coefficient of red luciferase through green filter
KGr Transmission coefficient of green luciferase through red filter
KRr Transmission coefficient of red luciferase through red filter
G Total luminescence from green luciferase
R Total luminescence from red luciferase

The following equations are derived from above and used in this calculator