Dual Luciferase Reporter Assay

Dual Luciferase Reporter Assay

What is a reporter assay?

The role of a reporter gene assay is to investigate the promoter of a gene of interest. Investigating the regulation of a gene's expression can be carried out by linking the promoter of interest to an easily detectable gene, such as the gene for firefly luciferase, which catalyses a reaction that produces light.

Usually, the cells are then exposed to conditions, or changes can be made in the order of the reporter, the effect of which can be easily tracked by measuring changes in light emission.

Essentially, a reporter assay uses a protein to measure a biological outcome using an observable parameter such as bioluminescence.

Dual Reporter Assays

Measuring two reporters in a single assay is called a dual-reporter assay or, if both reporters are luciferases, a dual-luciferase assay. The most commonly used dual-reporter assays measure both firefly and Renilla luciferase activities.

  • The Green Renilla luciferase is an intracellular protein with a greater serum stability as well as higher light output than native Renilla and Firefly luciferases. Consequently, Green Renilla reporter assays are more sensitive than assays employing native Renilla luciferase.
  • The Red Firefly luciferase is an intracellular protein with an emission peak shifted into the red spectrum compared to the native Firefly luciferase, which allows multiplexing with Gaussia, Cypridina and Green Renilla luciferases.

In a dual luciferase assay, the primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. The second marker can be used to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.

Assay Genie Dual Luciferase Reporter Assays

Product Code Product Name

MORV0192

Dual Luciferase Reporter Assay - Key Features

The Dual Luciferase Reporter Assay Kit sequentially measures the activity of Firefly and Renilla luciferases from the same sample. This kit is an excellent choice to detect changes in signalling pathways and gene regulation from cells transfected with reporter plasmids containing genes encoding Firefly and Renilla luciferases. In the assay, Firefly luciferase is detected with Luciferin as a substrate and then Renilla luciferase is detected with coelenterazine as a substrate. The Dual Luciferase Reporter Assay Kit can be used for the sensitive detection of the expression of Luciferase regulated by various gene elements.

Usually, the transcriptional regulatory element is cloned upstream of Firefly luciferase, or the 3'-UTR regulatory region is cloned downstream of firefly luciferase. The transfected cells are induced by a corresponding stimulator and lysed to determine the luciferase activity. The stimulatory-inducing effect of the regulatory elements is evaluated by luciferase activity. Renilla luciferase acts as an internal reference control for optimizing transfection efficiency and cell number. Firefly luciferase catalyzes the emission of Luciferin at 560 nm and Renilla luciferase catalyzes the emission of coelenterazine at 465 nm. This product is an economical alternative to other expensive Dual Luciferase assay kits.

Dual Luciferase Reporter Assay - Product Components

Component Amount

5 x Cell lysis Buffer

10 ml

Reaction Buffer II (Luciferase)

10 ml

Luciferase Substrate (Lyophilized)

1 vial

Stop & Reaction Buffer

10 ml

Renilla Substrate

200 µl

Dual Luciferase Reporter Assay - Protocol

1. Cell Lysis
Discard the cell culture medium and wash the cells twice with PBS. Add the appropriate amount of 1 × Cell Lysis Buffer as recommended in the table below. Stand still or shake for 5 min at room temperature, pipette up and down and transfer the cell lysate into a 1.5 ml centrifuge tube. Centrifuge for 2 min, 12000 × g at room temperature, and collect the supernatant for subsequent testing.

Cell Culture Plate 1 x Cell Lysis Buffer

6-well

500 µl

12-well

200 µl

24-well

100 µl

48-well

50 µl

96-well

20 µl

Note: If the expression level of luciferase is too low, the amount of Cell Lysis Buffer can be appropriately reduced to increase the protein concentration.

2. Firefly luciferase activity detection
Add 100 μl of Luciferase Substrate (which has been equilibrated to room temperature) to the detection tube or microplate. Carefully pipet 20 μl of the cell lysate into the test tube or the plate. Immediately after mixing rapidly, detect the Firefly luciferase reporter gene activity by a luminometer detector or a full-spectrum microplate reader.

3. Renilla luciferase activity detection
Add 100 μl of freshly prepared Renilla Substrate solution to the above reaction solution, and immediately after mixing rapidly, detect the renilla luciferase reporter gene activity by a luminometer detector or a full-spectrum microplate reader.