Dual Luciferin reporter gene assay

Luciferin reporter gene assay is a reporting system to detect the activity of Firefly Luciferase using luciferin as a substrate. Luciferase can catalyze the oxidation of luciferin to oxyluciferin. In the process of luciferin oxidation, bioluminescence will be emitted, which can be measured by fluorescence analyzer equipment. It is often used in the research of miRNA target gene verification and promoter transcriptive activity regulation. Dual luciferase usually refers to Firefly luciferase and Renilla luciferase. Renilla luciferase can oxidize coelenterazine in a non-ATP-dependent manner to produce fluorescence, and is often used as a reference in dual fluorescence detection.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Dual fluorescent reporter genes were constructed into different plasmids or the same plasmid.

2. The plasmid is transfected into the target cell.

3. The cells were washed with cold PBS after 1-day-culture, then 80-100 µL lysis buffer were added into the cells.

4. Passive lysised on ice for 30 min.

5. Centrifuge at 4 ℃ at 12000 rpm for 10 min.

6. Absorb 10 µL supernatant onto a 96-well plate dedicated to luciferase.

7. Add 50 µL Firefly Luciferase Buffer (FB) and 50 µL Renilla Luciferase Buffer (RB) in sequence.

8. Luminometer detects the fluorescence intensity at 550-570 nm and 480 nm.

For methods of gene editing and cultivation, please check the relevant protocols on our website.

1. A sufficient amount of substrate should be added to ensure the saturation of the substrate, otherwise it will cause a large deviation in the detection results.

2. Room temperature reaction. Each component of the reaction (cell lysis products, substrate working fluid, etc.) needs to be adjusted to room temperature.

3. The half-life of luciferase is generally about 30 minutes, and it can be detected immediately after adding the substrate, and it can be completed within 30 minutes as far as possible.

4. The substrate is sealed away from light, and the firefly luciferase substrate is stored at -20 ℃; The sea kidney luciferase substrate is recommended to be stored at -80 ℃.

5. The detection results of luciferase reporter gene experiment are very sensitive, and it is normal to have a certain difference in the value of multiple pores, and it is generally considered that the difference of the same order of magnitude is acceptable.