Transdetect double luciferase reporter assay kit

For transcription factor plasmids, the ORFs (open reading frames) of sterol regulatory element-binding protein 1 (SREBP1, XM_021624594.1), sterol regulatory element-binding protein 2 (SREBP2, XM_021625095.1), HNF1α (XM_021621309.1), CCAAT-enhancer-binding protein α (CEBPα, NM_001172496.1), CEBPβ (NM_001124447.1), CREB1 (XM_021597386.1), FOXO1 (XM_021618954.1), RXRα (XM_021590263.1), P65 (XM_021578194.1), and peroxisome proliferator-activated receptor γ (PPARγ, XM_021610844.1) were cloned into PCS2⁺ plasmids (Invitrogen, Carlsbad, CA, USA) by the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China).
HEK 293T cells were cultured according to the method described in the previous studies [12 (link)]. Transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the instructions. A total of 200 ng of promoter reporter plasmid, 600 ng of transcription factor plasmid, 20 ng of pRL-TK renilla luciferase, and 2.0 μL Lipofectamine 3000 were co-transfected in the 24-well plate in triplicate wells with three independent experiments. The TransDetect Double-Luciferase Reporter Assay Kit (TransGen, Beijing, China) and InfiniTE200 plate reader (Tecan, Männedorf, Switzerland) were used for the detection of dual luciferase activity.

The target genes of miR-183-5p and binding sites were examined with the biological prediction website Target Scan. Dual-luciferase reporter assay was employed to clarify whether there exists a targeting relationship between FOXO1 and miR-183-5p. Next, a pGL3-FOXO1 Wt reporter plasmid was constructed, which contained 3’UTR of FOXO1 that contained the potential miR-183-5p binding sites. The mutant form, pGL3-FOXO1 Mut, in which the potential miR-183-5p binding sites were mutated, was then constructed. The two reporter plasmids were respectively co-transfected into the HEK293 cells with over-expressed miR-183-5p plasmid and pRL-TK (internal reference plasmid expressing Renllia luciferase). Then, 24 h following transfection, the cells were lysed in accordance with the instruction of the TransDetect Double-Luciferase Reporter Assay Kit (FR201-01, Beijing TransGen Biotech Co., Beijing, China). Luciferase activity was detected using a dual-luciferase® reporter assay system (E1910, Promega Corporation, Madison, WI, USA). The relative luciferase activity was determined based on the ratio of firefly luciferase to Renilla luciferase. The experiment was conducted in triplicate.

The promoter of large yellow croaker SHP was cloned into the reporter plasmid (pGL3-basic vector, Promega, USA) by a ClonExpress II One Step Cloning Kit (Vazyme, China) (17 (link)). The expression plasmid of large yellow croaker NRF2 was stored in our laboratory. HEK 293T cells were seeded into 24-well plate (5 × 10⁵ cells) and co-transfected with reporter plasmids (200 ng/well), phRL-CMV plasmid (20ng/well, Promega, USA), and expression plasmids (200-600 ng/well). After 24 h transfection, cells were collected and the luciferase activity was measured using a TransDetect double-luciferase reporter assay kit (TransGen Biotech, China).

MiRwalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) was employed to predict the target genes of miR-9-5p. Leptin was found to be the target gene of miR-9-5p. The 3’UTR of leptin containing the miR-9-5p target site was cloned into the Sac I-Xba I site of the pmirGLO Vector (Promega, Madison, WI) to construct luciferase reporter plasmids. The primers used for plasmid construction are listed in Table 1. Multiple cloning sites were located downstream of firefly luciferase gene. HeLa cells were seeded into 24-well plates in triplicate. Then, the pmirGLO-leptin-3′ UTR of wild or mutant type was co-transfected with the synthetic miR-9-5p mimic into HeLa cells after the cell density reached 70–80%. Firefly luciferase (luc2) activity was measured 48 h after transfection and normalized to Renilla luciferase activity according to the TransDetect® Double Luciferase Reporter Assay Kit instructions (Transgen, Beijing, China).

A website (starbase)¹ was used to predict whether miR-23a could target MDM2. The dual luciferase reporter gene assay was used to verify whether there was a binding site between miR-23a and MDM2. The pGL3-MDM2 wild type (Wt) and pGL3-MDM2 mutant type (Mut) were co-transfected with miR-23a mimic/mimic-NC and pRL-TK (internal reference plasmids expressing renilla luciferase) into HEK293 cells. After 24 h of transfection, the cells were lysed according to the steps of TransDetect Double-Luciferase Reporter Assay Kit (FR201-01, TransGen Biotech, Beijing, China) and the supernatant was collected. The dual-luciferase reporter assay system (E1910, Promega) was used to detect luciferase activity. The relative luciferase activity of firefly luciferase/renilla luciferase was used as the relative luciferase activity. Similarly, pGL3-NEAT1 Wt and pGL3-NEAT1 Mut were constructed, and then luciferase activity was detected in the same way for verification on binding between lncRNA NEAT1 and miR-23a.