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Double luciferase reporter analysis system

Manufactured by Promega
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The Double Luciferase Reporter Analysis System is a tool used for gene expression analysis. It allows simultaneous measurement of firefly and Renilla luciferase activities in the same sample, enabling normalization of experimental results.

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Luciferase reporter analysis system Luciferase reporter system

3 protocols using double luciferase reporter analysis system

1

Luciferase Assay for miRNA-497-5p Regulation

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For the luciferase reporter gene assay, 5 × 105 HEK293T cells were inoculated in a 24-well plate overnight. pmirGLO-PDCD4-WT or pmirGLO-lncRNA XIST-WT reporter plasmids (150 ng each) and their mutant vectors were cotransfected into cells with 50 nM mimic of miRNA-497-5p using Lipofectamine 2000 reagent. After 36 h of cell culture, the firefly and Renilla luciferase activities were determined by a double Luciferase Reporter Analysis System (Promega) based on the manufacturer’s instruction manual. The relative luciferase activity was calculated based on the firefly/Renilla fluorescence ratio.
Zhang Y., Zhu Z., Huang S., Zhao Q., Huang C., Tang Y., Sun C., Zhang Z., Wang L., Chen H., Chen M., Ju W, & He X. (2019). lncRNA XIST regulates proliferation and migration of hepatocellular carcinoma cells by acting as miR-497-5p molecular sponge and targeting PDCD4. Cancer Cell International, 19, 198.
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2

Validating miR-489 Regulation of SPIN1

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SPIN1 3′UTR containing miR-489 target binding sites of wild-type or mutant sequences were cloned into a carrier of luciferase reporter, which was transfected with miR-489 mimics in H9C2 cells. Then, luciferase activity was determined by a double luciferase reporter analysis system (PROMEGA).
Li W., Zhang Y., Wang J., Li Q., Zhao D., Tang B., Wang S, & Shao H. (2022). MicroRNA-489 Promotes the Apoptosis of Cardiac Muscle Cells in Myocardial Ischemia-Reperfusion Based on Smart Healthcare. Journal of Healthcare Engineering, 2022, 2538769.
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3

Characterization of miRNA-613 3'UTR

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The expression vectors of wild-type miRNA-613 3′UTR and mutant miRNA-613 3′UTR were successfully constructed first, which was operated according to the instructions of the luciferase detection kit (Sangon Biotech, Shanghai), and the detection results were observed after 48 h with the double luciferase reporter analysis system (PROMEGA) and counted.
Zhou D., Wang Y., Hu H., Liu H., Deng J., Li L, & Zheng C. (2022). lncRNA MALAT1 promotes HCC metastasis through the peripheral vascular infiltration via miRNA-613: a primary study using contrast ultrasound. World Journal of Surgical Oncology, 20, 203.
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