Dual glo luciferase reporter system

The 3′-untranslated region (UTR) of human CCND1 was amplified from human genomic DNA and individually inserted into the pmiR-RB-REPORT™ (Ribobio, Guangzhou, China) using the XhoI and NotI sites. Similarly, the fragment of CCND1 3′-UTR mutant was inserted into the pmiR-RB-REPORT™ control vector at the same sites. For reporter assays, A549 cells were co-transfected with wild-type (mutant) reporter plasmid and miR-134 mimics (miR mimic NC) using Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were measured in cell lysates using the Dual-Luciferase Reporter Assay system. Luciferase activity was measured forty-eight hours post-transfection using dual-glo luciferase reporter system according to the manufacturer's instructions (Promega, Madison, WI, USA). Firefly luciferase units were normalized against Renilla luciferase units to control for transfection efficiency.

The 3'-untranslated region (UTR) of human EGFR was amplified from human genomic DNA and individually inserted into the pmiR-RB-REPORT TM (Ribobio, Guangzhou, China) using the XhoI and NotI sites. Similarly, the fragment of EGFR 3'-UTR mutant was inserted into the pmiR-RB-REPORT TM control vector at the same sites. For reporter assays, HCT116 cells were co-transfected with wild-type (mutant) reporter plasmid and miR-875-5p mimics (miR mimic NC) using Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were measured in cell lysates using the Dual-Luciferase Reporter Assay system. Luciferase activity was measured forty-eight hours post-transfection using dual-glo luciferase reporter system according to the manufacturer's instructions (Promega, Madison, WI, USA). Firefly luciferase units were normalized against Renilla luciferase units to control for transfection efficiency.

The wild-type RP11-142A22.4 sequence was cloned into the pmiR-RB-Report vector (RiboBio Co., Guangzhou, China), and mutants were generated using site-directed mutagenesis as described above. The mutations were confirmed by sequencing, with vectors containing a mutant sequence used as a control. Preadipocytes were seeded in 96-well plates at a density of 4 × 10³ cells per well 24 h before transfection. The cells were then transfected with the wild-type or a mutated reporter vector, and lysates were obtained 24 h post-transfection. The dual-luciferase assay was performed using the Dual-Glo Luciferase Reporter System (Promega, Madison, WI) according to the manufacturer’s protocols.

To generate the luciferase reporter plasmids, a single perfectly complementary microRNA target sequence was inserted into the XhoI site in the 3′ UTR of the Renilla luciferase gene in the psi-check-2 dual luciferase reporter construct (Promega). The firefly luciferase gene, also present in the plasmid, was used as a transfection control. 500 ng of plasmid was transiently transfected into 1×10⁶ S2-DRSC cells/ml in a 96 well plate using effectene (Qiagen). 48 hours later the cells were lysed using 40 ul 1× passive lysis buffer and subjected to the Dual-glo luciferase reporter system (Promega) and analysed using the MicroLumatPlus LB96V Microplate Luminometer. Three independent replicates were carried out on different days. The reported repression values are calculated as the ratio of Renilla luciferase to firefly luciferase activity, normalized to that of the empty reporter construct for each day of transfection. Raw values from the luminometer are shown in Table S3. A paired t-test was used to determine if suppression of the target reporter differed significantly from the empty reporter construct at a 0.05 significance level for each microRNA target site. Statistical analysis was carried out using the R software (R Development Core Team).

The 3′-UTR of human E2F3 or lncRNA NEAT1 were amplified from human genomic DNA and individually inserted into the pmiR-RB-REPORT™ (Ribobio, Guangzhou, China) using the XhoI and NotI sites. Similarly, the fragment of E2F3 or lncRNA NEAT1 3′-UTR mutant was inserted into the pmiR-RB-REPORT™ control vector at the same sites. For reporter assays, A549 and H1299 cells were co-transfected with wild-type (mutant) reporter plasmid and miR-Ribo™ mimics (miR-Ribo™ negative control) using Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities were measured in cell lysates using the Dual-Luciferase Reporter Assay system. Luciferase activity was measured forty eight hours post-transfection using dual-glo luciferase reporter system according to the manufacturer's instructions (Promega, Madison, WI, USA). Firefly luciferase units were normalized against Renilla luciferase units to control for transfection efficiency.

A wild-type circSAMD4A sequence was cloned into a pmiR-RB-Report vector (Ribobio Co., Guangzhou, China), while simultaneously generating mutants using site-directed mutagenesis as described above. The mutations were confirmed via sequencing with vectors containing a mutation sequence used as a negative control. Preadipocytes were seeded in 96-well plates at a density of 4 × 10³ cells per well 24 h before transfection. The cells were then transfected with either the wild-type or mutated reporter vectors with lysates obtained at 24 h post-transfection. The Dual-Glo Luciferase Reporter System (Promega, Madison, WI) was used to perform the dual-luciferase assay according to the manufacturer's protocols.

The plasmids used for firefly luciferase reporter assay were packaged by Genechem (Shanghai Genechem, China). And the plasmids designated IGF1R-WT, TGFBRI-WT (wild-type of miR-140-5p, targeting to IGF1R 3′-UTR and TGFBRI 3′-UTR), IGF1R-MU and TGFBRI-MU (mutated miR-140-5p, targeting to IGF1R 3′-UTR and TGFBRI 3′-UTR) were used. The mimic and the mimic negative control of miR-140-5p were purchased from Ribobio (Guangzhou RiboBio, Guangzhou, China). HEK293 cells were cultured in complete medium for 24 h before transfection. 0.05 μg firefly luciferase reporter, 0.05 μg IGF1R/TGFBRI plasmid, and 0.01 μg Renilla luciferase control vector were co-transfected into HEK293 cells by lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. At 48 h post-transfection, luciferase activity was detected by using the Dual-Glo luciferase reporter system (Promega Corp., Madison, WI, USA) in accordance with the protocols of manufacturer [20 (link)]. The relative luciferase activity value was achieved against the renilla control.