Luc pair duo luciferase assay kit 2

The plasmid containing FoxM1 3′-UTR for the putative miR-320d binding and luciferase reporter gene was constructed by using pEZX vector (GeneCopeia™). Two mutants of FoxM1 3′-UTR were constructed by the GeneCopeia™ company. The oligonucleotide sequences for FoxM1 3′-UTR mutated types and wild type were listed in Additional file 1: Table S2. Then OE-19 cells were transfected with constructed plasmids by lipofectamine™ 2000 (Invitrogen, USA), and group settings were shown as followed (Additional file 1: Table S3, n = 5 for each group): (1) negative control, cells transfected with NC plasmid and wild FoxM1 3′-UTR plasmid; (2) positive control, cells transfected with stable expression of miR-320d plasmid and wild type FoxM1 3oxM1 plasmid; (3) cells transfected with stable expression of miR-320d plasmid and mutant site 1 of FoxM1 3′-UTR plasmid; (4) cells transfected with stable expression of miR-320d plasmid and mutant site 2 of FoxM1 3′-UTR plasmid; (5) cells transfected with stable expression of miR-320d plasmid and mutant site 1 and 2 of FoxM1 3′-UTR plasmid. After the transfection, cells were harvested and analyzed for luciferase activity using Luc-Pair ™ Duo-Luciferase Assay Kit 2.0 (GeneCopeia™).

For the promoter activity assays, ICPs were cotransfected with reporter plasmid and MYOD1 over-expression vector or control vector. The TK-Renilla reporter was also cotransfected to each sample as an internal control using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) in 48-well plates. The miRNA target verification assay was also performed in ICPs. Wild-type or mutant KLF4-3′-UTR dual-luciferase reporter (200 ng) and miR-206 mimic or NC mimic (50 nmol/L) were cotransfected into ICPs. After 48 h transfection, cells were washed by PBS twice, and the activities of Firefly and Renilla luciferase were measured according to the manual of Luc-pair Duo-Luciferase Assay Kit 2.0 (GeneCopoeia, Rockville, MD, USA). All the data were acquired by averaging the results from three independent repeats.

A luciferase miRNA 3′-UTR target vector carrying the predicted miR-152 binding site in the TFRC 3′-UTR, miRNA 3′-UTR target vector harboring two base mutations in the predicted miR-152 binding site in the TFRC 3′-UTR, and miRNA 3′-UTR target control vector were constructed and purchased from GeneCopoeia Inc. (Rockville, MD). To assess the direct interaction between miR-152 and the TFRC 3′-UTR, the luciferase constructs (1 μg) and the 80 nM of miR-152 mimic (Life Technologies) were co-transfected into SK-HEP1 cells, which were seeded in 6-well plates (1 × 10⁶ cells/transfection) using Lipofectamin™ 3000 transfection reagent (Life Technologies) following the manufacturer's recommendations. SK-HEP1 cells transfected with miR™ Negative Control #1 (Life Technologies) served as the control. Forty-eight hours after the transfection, cells were harvested by mild trypsinization, washed in PBS and the luciferase activity was measured using a Luc-Pair Duo-Luciferase Assay Kit 2.0 (GeneCopoeia Inc.) according to the manufacturer's instructions.

Wild-type and mutant RPS6KA2 enhancer luciferase reporter plasmid were constructed by cloning the 2000 bp fragment before TSS into pEZX-FR01 plasmid. MIR548D1 promoter luciferase reporter plasmid was constructed by cloning the 1000 bp fragment before TSS into pEZX-FR01 plasmid. The 747 bp wild-type and mutant 3′UTR region of JUND gene were cloned into pEZX-MT06 plasmid (Genecopoeia, Guangzhou, China). Cells were seeded in 60 mm dishes and transfected with mentioned plasmids using FuGene 6 transfection reagent (Roche) for 24 h. Cell lysates were extracted and luciferase activity was measured using the Luc-Pair™ Duo-Luciferase Assay Kit 2.0 (Genecopoeia, Guangzhou, China). All experiments were performed three times in triplicate. Relative luciferase activities were calculated as fold induction compared with vector control.

The promoter sequence of Cdh1 was cloned into pEZX-FR01 (GeneCopoeia) before the Firefly luciferase (Fluc), the Renilla luciferase (Rluc) was used as tracer gene. mESCs were planted in 24-well plates at 2 × 10⁴ per well, and the Tfap2c or control vector (0.5 µg) and pEZX-FR01 (100 ng) were cotransfected into the cells with Lipofectamine Stem Transfection Reagent (Invitrogen, STEM00015). At 48 h after transfection, the luciferase activity was detected according to the instructions for the Luc-Pair Duo-Luciferase Assay Kit 2.0 (GeneCopoeia).