Biolux gaussia

PC3 cells were cultured in a 6-well plate and transfected with miR-155-5p mimic and miR-155-5p negative control (NC; Shanghai GenePharma Co., Ltd.) when the cells were 70% confluent. After 48 h, RT-qPCR was performed to evaluate transfection efficiency. SPOCK1 expression was evaluated using RT-qPCR and western blot assays to verify whether SPOCK1 is a direct target gene of miR-155-5p.
For the luciferase reporter assay, PC3 cells were seeded into 96-well plates at a density of 2×10⁴ cells/well and grown to 70% confluence. Cells were co-transfected with miR-155-5p mimic or miR155-5p NC for 48 h at 37°C, and SPOCK1-3′-untranslated region (UTR)-wild-type (WT) or SPOCK1-3′-UTR-mutant (MUT) plasmids (Shanghai GenePharma Co., Ltd.) using Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.). A luciferase assay kit (BioLux^® Gaussia; New England BioLabs, Inc.) was used to evaluate luciferase activity according to the manufacturer's protocol.

Bioinformatics analysis with TargetScan, and luciferase reporter assays were conducted to verify that SOX17 was a direct target gene of miR-194-5p. For luciferase reporter assays, MCF-7 cells were seeded into 96-well plates at a density of 2×10⁵ cells/well and grown to 70% confluence. Following the implementation of site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis kit (Guangzhou RiboBio Biotechnology Co., Ltd.), cells were co-transfected with miR-194-5p mimic or miR-194-5p NC for 48 h at 37°C (50 ng; Guangzhou RiboBio Biotechnology Co., Ltd.), and SOX17-3' untranslated region (UTR)-wild-type (WT) (50 ng) or SOX17-3'UTR-mutant (MUT) (50 ng) plasmids (Guangzhou RiboBio Biotechnology Co., Ltd.) using Lipo6000™ Transfection Reagent (0.2 µl). Furthermore, transfection efficiency was normalized to a Renilla luciferase vector (pRL-CMV; Promega Corporation). The luciferase assay kit (BioLux^®Gaussia; New England Biolabs, Inc., Ipswich, MA, USA) was used to evaluate lucif-erase activity according to the manufacturer's protocol.