Luc pair duo luciferase ht assay kit

The combination between the circLRRFIP1 and miR-15 family was detected by the Dual-luciferase assay. The wild type (pmirGLO-WT) and mutated sequences (pmirGLO-MT) of circLRRFIP1 and AKT3 (NCBI Reference Sequence: XM_046914424.1) which harbor the conserved binding site for the miR-15 family members were synthesized and cloned into the pmirGLO Dual-Luciferase reporter vector (Sangon Biotech, Shanghai, China). DF-1 cells were plated in a 48-well plate and transfected with the miR-15 family members or mimic-NC along with the appropriate luciferase reporter vectors. The Luc-Pair Duo-Luciferase HT Assay Kit (GeneCopoeia, Rockville, MD) and a microplate reader (US BioTek Laboratories, Shoreline, WA) were used to measure the relative luciferase activity. Then the fluorescence intensity of the firefly luciferase was compared with the Rinilla luciferase, and also compared with it in different treatments.

HepG2 cells were co-transfected with ABCA1 renilla reporter plasmid and TK-control firefly luciferase vector at 20:1 using Lipofectamine 3000 reagent according to manufacturer’s instructions for 24hrs. Afterwards, 7000 cells were seeded in to 384 well f-bottom, white plates (Greiner Bio-One). Test compounds were added to the plates in a final working volume of 30 µL (for a final concentration of 50 µM) in presence of DMSO or 1 µM GW3965 and incubated for 20–24hrs at 37°C. Luciferase activity was measured using the Luc-Pair Duo-Luciferase HT Assay kit (GeneCopoeia, Rockville, MD, USA) by adding luciferase substrates sequentially following manufacturer instructions. Luminescence was measured with 2 s integration times in a microplate reader (BioTek Cytation 5), 15 min after adding each substrate. The addition of reagents to the microplates was timed at each step to match the reading time delays and reading sequence of the microplate reader.