Renilla luciferase thymidine kinase prl tk plasmid

Luciferase reporter assays were performed using the psiCHECK2-3'UTR vector (Addgene, Inc.). Prior to the DLRA, the targeted 3'untranslated region (3'UTR) of EMMPRIN was amplified using PCR, and the product was inserted into the psiCHECK2-3'UTR vector, which were later examined through DNA sequencing. In a 24-well plate, U87 cells were transfected with the wild-type reporter plasmid, the Renilla luciferase-thymidine kinase (pRL-TK) plasmid (Promega Corporation), and miR-124 mimic, a miR-124 inhibitor (miRNA inhibitor oligonucleotide against miR-124; 5'-GGCAUUCACCGCGUGCCUUA-3), or negative control (NC) using Lipofectamine^® RNAiMAX reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 24 h, the luciferase activity was detected by the luciferase reporter assay using the Dual Luciferase Assay system (Promega Corporation). Renilla luciferase activity was normalized to firefly luciferase activity. Cell lysates were subjected to luciferase activity measurement according to the manufacturer's instructions.