Luciferase reporter assay is a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase. Luciferase can catalyze the oxidation of luciferin into oxyluciferin. During the oxidation of luciferin, bioluminescence is emitted. The bioluminescence released during the oxidation of luciferin can be measured by equipment such as microplate readers. It is often used in the verification of miRNA target genes and the regulation of promoter transcription activity.
1. Experimental principle
Using the characteristics of luciferase and substrate to produce chemiluminescent reaction, the regulatory elements of target gene transcription are cloned upstream/downstream of the firefly luciferase gene to construct a luciferase reporter plasmid. Then transfect the cells, and lyse the cells after appropriate stimulation or treatment to measure the luciferase activity. The effect of different stimulations on the regulatory elements of interest before and after stimulation can be judged by the level of luciferase activity. Dual luciferase usually refers to firefly luciferase and Renilla luciferase. Since single reporter gene experiments are often affected by various experimental conditions, dual reporter genes provide a baseline for the experiment through the co-transfection "control" as an internal reference, thereby minimizing the impact of external factors such as cell activity and transfection efficiency on the experiment, making the data results more reliable.
2. Experimental principle diagram
2.1 Interaction between miRNA and target gene mRNA
2.2 Interaction between transcription factors and promoters
3. Experimental steps
3.1 Sample preparation (different groups are designed according to different experimental purposes. This experiment takes miRNA target gene verification as an example)
3.1.1 Cells are seeded into a 12-well cell culture plate at a density of 50%.
3.1.2 When the cells grow to about 70%, the luciferase reporter gene plasmid is transfected. Three replicate wells are set for each sample, and the groups are divided into 4 groups: empty + NC group, empty + miRNA mimics group, recombinant plasmid + NC group, and recombinant plasmid + miRNA mimics group.
3.1.3 Preparation of plasmid group: 50 ng plasmid per well, and 20 μL serum-free medium per well are calculated, marked as A.
3.1.4 Preparation of miRNA NC/mimics: The final concentration of miRNA NC/mimics is 20 nM, and the required amount is calculated by 20 μL serum-free medium per well, marked as B and C respectively.
3.1.5 Prepare the corresponding transfection reagent: calculate the required amount according to 0.5 μL transfection reagent per well and 20 μL serum-free medium per well, marked as D.
3.1.6 Incubate the four diluted reagents at room temperature for 5 min.
3.1.7 Mix the diluted plasmid DNA and miRNA mimics with the corresponding transfection reagents respectively and incubate at room temperature for 20 min.
3.1.8 Add the reagents in step 3.1.7 to the wells.
3.1.9 After 6 h of transfection, replace with fresh complete medium.
3.2 Dual reporter gene detection
3.2.1 After 36-48 h of plasmid co-transfection, discard the medium and wash the cells with 100 μL 1×PBS.
3.2.2 Tilt the 12-well plate and absorb the remaining PBS.
3.2.3 Dilute 5×PLB (lysis buffer) into 1×PLB with deionized water (prepared and used immediately), and place at room temperature before use.
3.2.4 Add 50 μL of diluted 1×PLB to each well, and shake on a shaker for 20-30 min to ensure that the lysis buffer completely lyses the cells.
3.2.5 Add 10 μL of the supernatant in step 3.2.4 to each well of a white opaque 96-well ELISA plate, add 100 μL of the pre-mixed Luciferase Assay Reagent II, measure the data after 2 s, and detect the luciferase reaction intensity.
3.2.6 After the determination, add 100 μL of the pre-mixed Stop&Glo Reagent to each well, let it rest for 2 s, measure the data, and detect the internal reference Renilla luciferase reaction intensity.
3.2.7 Record the readings. Each sample will have 3 values: RLU1-firefly luciferase reaction intensity, RLU2-internal reference Renilla luciferase reaction intensity, calculate the ratio of the two sets of data, i.e. RLU1/RLU2.
4. Result example
In this result, the luciferase activity of XXXC5-WT and XXXK4-WT was reduced after treatment with miRNA-xxx-5p, indicating that they are target genes of miMRNA-xxx-5.
References
[1] Lomakina GY, Modestova YA, Ugarova NN. Bioluminescence assay for cell viability. Biochemistry (Mosc). 2015 Jun;80(6):701-13.
[2] Nieuwenhuijsen BW, Huang Y, Wang Y, Ramirez F, Kalgaonkar G, Young KH. A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions. J Biomol Screen. 2003 Dec;8(6):676-84.
[3] Xu YZ, Kanagaratham C, Jancik S, Radzioch D. Promoter deletion analysis using a dual-luciferase reporter system. Methods Mol Biol. 2013;977:79-93.
[4] Ghazawi I, Cutler SJ, Low P, Mellick AS, Ralph SJ. Inhibitory effects associated with use of modified Photinus pyralis and Renilla reniformis luciferase vectors in dual reporter assays and implications for analysis of ISGs. J Interferon Cytokine Res. 2005 Feb;25(2):92-102.