Principles of the dual luciferase experimentIt is to use the characteristics of luciferase and substrate binding to chemiluminescence reaction, and cloning the regulatory element of gene transcription in the upstream of firefly luciferase gene to construct a luciferase reporter plasmid. Cells are then transfected, and cells are lysed after appropriate stimulation or treatment, and luciferase activity is measured. The effect of different stimuli on the regulatory element of interest before and after stimulation or by the level of luciferase activity can be determined. At the same time, in order to reduce the influence of intrinsic variable factors on the accuracy of the experiment, the plasmid with rinilla luciferase gene was used as the control plasmid to co-transfect the cells with the reporter plasmid to provide an internal control for transcriptional viability, so that the test results were not interfered by the change of experimental conditions.
Luminescence principle. Specific steps for the double luciferase experiment.
1. Construction of reporter plasmids. Insert the fragment of interest into a luciferase-expressing reporter vector, such as PGL3-BASIC.
2. Transfect cells. Cells were co-transfected with a reporter plasmid and PHRL-TK (internal control) and treated as needed. When co-transfected, the reporter plasmid:reference transfection amount is typically 10:1 to 50:1 due to the strong promoter of the internal control.
Luciferase Activity Assay:For initial use, prepare LAR II, a substrate for Firefly Luciferase. Dissolve LAR II in LAR II buffer and aliquot -80 Store protected from light. Add 1x PLB and lyse the cells for 15 min at room temperature. Formulate STOP&GLO, a substrate for Renilla Luciferase, capable of terminating the LAR II reaction. Determination of fluorescence values. Add 10 μL of cell lysate to 40 μL of LAR II, pipette and mix, and test the reading, which is the value of Firefly Luciferase. Add 40 ul STOP&GL and read it again, which is the value of Renilla Luciferase. Data processing. First, the ratio of firefly luciferase renillaluciferase in each tube was calculated, and then the ratio of the control group was used as a unit of 1 to obtain the relative luciferase activity of different treatment groups, that is, the regulatory activity of gene transcription in the treatment group. Interpretation of results
Literature I：pmid: 28697764；if=41.444
(1) Method
(2) Results
Verify the binding of LINC00673 and MIR-150-5P. Wild-type LINC00673 binds to mir-150-5p, but mutant LINC00673 cannot. Luciferase was linked to the Linc00673 gene by constructing a luciferase reporter vector, and then transfected into cells to detect fluorescent activity. The results show that:mirpMIMICS significantly reduced wild-typelinc00673luciferase activity, but for mutant formslinc00673Luciferase activity had no effect。The above results show that:linc00673Available with mirp-combined
Literature II：pmid: 35963157；if=5.741
(1) Method
(2) Results
The binding of YAF2 and MIR-217-5P was verified. Wild-type YAF2 binds to Mir-217-5P, but mutant YAF2 cannot. Luciferase was linked to the YAF2 gene by constructing a luciferase reporter vector and then transfected into cells for detection. The results show that:mirpMIMICS significantly reduced the luciferase activity of wild-type YAF2, but had no effect on the luciferase activity of mutant YAF2。The above results show that:yaf2 is available with mirp-combined
Literature III：pmid: 35212607；if=6.832
(1) Method
(2) Results
In this study, the binding of PEG10 and Mir-449A and RPS2 to Mir-449A was verified. Here we take PEG10 and Mir-449A as examples. Wild-type PEG10 binds to Mir-449A, but mutant PEG10 cannot. Luciferase is linked to the PEG10 gene by constructing a luciferase reporter vector and then transfected into cells for detection. The results show that:mir-449aMIMICS significantly reduced wild-typepeg10luciferase activity, but for mutant formspeg10Luciferase activity had no effect。Proofpeg10withmir-449aTargeted binding
Literature IV：pmid: 35110549；if=9.685
(1) Method
(2) Results
Verify the binding of PAARH and HOTTIP to six miRNAs, respectively. The above results showed that the fluorescence activity of the miRNA transfection group was significantly reduced, indicating that there was targeted binding between miRNA and Paarh and Hottip.
Note that there were no mutant results in this study, and there are very few such examplesGenerally, luciferase results need to include mutant type and wild-type, so pay attention to it when designing your own experiment
Fluorescence