Dual luciferase reporter gene assay kit

The dual-luciferase vector pmirGLO was purchased from Promega (Promega, cat. C838A). The 3ʹ UTR of PDGFC was amplified by PCR using genomic DNA from 293 T cells as a template. A clone with a sequence that was identical to the NCBI reference sequence NM_016205 was cloned into the pmirGLO vector at the SacI and SalI restriction sites. Two putative m⁶A recognition sites were identified in the 3ʹ UTR. Mutagenesis from A to T was generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent, USA) according to the manufacturer’s instructions. Luciferase activity was measured with a Dual-Luciferase Reporter Gene Assay Kit (Promega, cat. E1910). The activity of firefly luciferase was normalized to that of Renilla luciferase to evaluate reporter expression efficiency.

MiRNAs targets were predicted using the algorithms TargetSan (https://www.targetscan.org). The Bcl-2 gene was input and then a list of miRNAs that target certain base pairs on Bcl-2 were shown in the map. The levels of these miRNAs in our experimental conditions were then examined. Only those that had a significant change by UUO with/without IGF-1 were used as candidates for further analyses. Luciferase-reporters were constructed using molecular cloning technology. Target sequences for Bcl-2 3′-UTR and Bcl-2 3′-UTR with a site mutation at miR-429 binding site (Bcl-2 3′-UTR mut) were purchased from Creative Biogene (Shirley, NY, USA). Purified renal epithelial cells were seeded in 24-well plates for 24 hours, after which they were co-transfected with 1 μg of Luciferase-reporter plasmids and miR-429-modified plasmids. Luciferase activities were evaluated using the dual-luciferase reporter gene assay kit (Promega, Beijing, China).

The relationship between circ_0000790 and miR-374c, miR-374c, and FOXC1 was analyzed using a biological prediction website (https://cm.jefferson.edu/rna22/Interactive/), and the dual-luciferase reporter gene assay was employed to further verify whether circ_0000790 or FOXC1 was the target gene of miR-374c. According to the predicted binding site, the artificially synthesized mRNA 3′ UTR fragments of circ_0000790 and FOXC1 were inserted into plasmid pGL3-basic (P2129, Shanghai Hewu Biotechnology, Shanghai, China). The complementary sequence mutation site of the seed sequence was designed on FOXC1-wild-type and constructed into the reporter plasmids, which were comprised of a combination of Luc-circ_0000790-wild-type, Luc-circ_0000790-mut, FOXC1-wild-type, and FOXC1-mut, respectively. The correctly sequenced luciferase reporter plasmids were co-transfected into HEK293T cells with miR-374c and NC, respectively. The luciferase activity was measured using the dual-luciferase reporter gene assay kit (E1910, Promega, Madison, WI, USA). Fluorescence intensity was measured using a GloMax 20/20 luminometer fluorescence detector (E5311, Shaanxi Zhongmei Biotechnology, Shanxi, China). The experiment was repeated three times independently.

After transfection 24 h, according to the manufacturer’s instructions, luciferase reporter analyses were performed using a dual-luciferase reporter gene assay kit (Promega, Madison, WI, USA), and Firefly and Renilla luciferase activity were measured. The 293T cells were removed from growth media and lightly washed in 1× PBS. After the culture plate added 100 μL Passive Lysis Buffer (PLB) and then shaken at room temperature for 15 min, the solution was transferred to the PCR tube, centrifuged by 12,000 rpm for 5 min, and collected the supernatant. Add the collected supernatant at 5 μL per hole in the enzyme plate, then add 50 μL luciferase assay reagent II (LARII) to each well, gently shake and mix, and detect the fluorescence value of Firefly luciferase. Then 50 μL of Stop&Glo, was added to each well to detect the fluorescence value of Renilla luciferase. The relative Firefly luciferase activity was calculated by normalizing to Renilla luciferase activity. All experiments were performed in three independent biological replications, and reactions of each sample were carried out in triplicate. All data were expressed as means ± the SEM and determined by one-way ANOVA. p values < 0.05 were considered statistically significant.

Wild-type and mutant UPF1 were constructed and then co-transfected with tRF-Leu-AAG mimic/negative control and wild-type/mutant UPF1 into cells. Dual-Luciferase Reporter gene assay kit (Promega, Madison, WI, USA) was used to detect the activity of cell luciferase.

Online databases (http://www.microrna.org/; http://mirdb.org/index.html) were used to predict the putative binding site of miR-873 in 3′-UTR of ABCA1. U251 cells were transiently cotransfected with a luciferase reporter plasmid encoding the 3′-untranslated region (3′-UTR) of ABCA1 (pMIR-Report-ABCA1) and the pcDNA-3.1(-)-miR-873 vector or an empty vector. A dual-luciferase reporter assay system was used to detect the functional regulation of ABCA1 mRNA by miR-873. The luciferase activity was quantified 36 h after transfection using the Dual- Luciferase Reporter Gene Assay Kit (Promega, Madison, WI, USA) and a luminometer system (Tecan Spark™ 10M, Tecan, Switzerland). The firefly luciferase activity was normalized with the Renilla luciferase activity to control for the transfection efficiency.