Luciferase activity was assessed according to the Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI) using a Veritas™ 96-well Microplate Luminometer (Promega, Madison, WI) with substrate dispenser (Promega, Madison, WI). HEK293T cells transduced with leti-miR-424 or control virus were seeded in 96-well plates with 70% confluence. 12 hours later, the cells were cotransfected with 50 ng pGL3-Promoter -UTR and 10 ng pRLTK using the Lipofectamine LTX. After 24 hours of transfection, the cells were harvested for firefly and Renilla luciferase activity assay. The renilla luciferase activities were used to normalize the transfection efficiency.
Substrate dispenser
Manufactured by Promega
Sourced in United States
The Substrate Dispenser is a versatile laboratory equipment designed to accurately and efficiently dispense liquids or suspensions. It features user-friendly controls and can be programmed to deliver precise volumes, making it a valuable tool for a wide range of applications in various research and diagnostic settings.
Automatically generated - may contain errors
2 protocols using substrate dispenser
1
Dual-Luciferase Assay for miRNA Validation
2
Luciferase Assay for miR-379-3p Targeting
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To generate the luciferase construct, the circPOLQ and IL-10 genes containing the miR-379–3 p binding site were synthesized by GeneChem (China) and inserted into the GV272 vector. Next, PMA-treated THP-1 cells were treated with IL-10-3′-UTR-Luc firefly luciferase constructs (wild-type or mutant) using Lipofectamine 3000 reagent (Invitrogen, USA). The Luc firefly luciferase construct (wild-type or mutant) was co-transfected with an miR-379–3 p mimic. Finally, 48 hours after transfection, the cell lysates were collected, and the firefly/Renilla luminescence was quantified using a Veritas 96-well microplate luminometer (Promega, USA) following the procedure of the Dual-Luciferase Reporter Assay Kit procedure (Biyuntian, China). Neutase activity was assessed using a substrate dispenser from Promega (USA). Renilla luciferase activity was normalized to firefly luciferase activity.
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