To demonstrate the functional interaction between two molecules, the sequence containing the wild type binding sites (WT) or the sequence with mutated binding sites (MUT) were cloned into the PmirGLO firefly luciferase reporter vector (Promega). The reporter plasmid and Renilla luciferase (hRlucneo) control plasmid were co‐transfected into cells with either miRNA mimic or miR‐NC. 48 h after the transfection, the relative luciferase activities were measured using Dual‐Luciferase Reporter Assay Kit (Promega) on a luminescence microplate reader (Biorad).
Pmirglo firefly luciferase reporter vector
Manufactured by Promega
Sourced in United States
The PmirGLO firefly luciferase reporter vector is a plasmid that contains the firefly luciferase gene as a reporter. The luciferase gene is driven by a promoter, allowing for the quantification of promoter activity in transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Luminescence microplate reader, by Bio-Rad (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Penicillin, by GE Healthcare (1 mentions)
Streptomycin, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Quikchange multi site directed mutagenesis kit, by Agilent Technologies (1 mentions)
5 protocols using pmirglo firefly luciferase reporter vector
1
Luciferase Assay for miRNA Binding
2
Dual-Luciferase Reporter Assay Protocol
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The predicted interacting sites (WT) or the mutated sequences (MUT) were cloned into the pmirGLO firefly luciferase reporter vector (Promega Corp., Madison, USA). The cells at a logarithmic phase were seeded in the 24-well plates at a density of 1.5 × 104/well. 1 μg WT or MUT reporter vector and 1 μg pRL Renilla control vector were introducted into CRC cells using the LipofectamineTM 2000 reagent. After 48 h of transfection, a dual-luciferase reporter assay system (Promega) was utilized to detect firefly luciferase activity from the reporter vector and renilla luciferase activity from the control vector, respectively.
3
Luciferase Reporter Assay for miR-142-3p Targeting
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The 3′-UTR regions of AC9 containing the predicted miR-142-3p specific binding sites were amplified by RT-PCR and cloned into the pmirGLO firefly luciferase reporter vector (Promega Corporation) to obtain the wild-type luciferase reporter plasmids (wt-AC9). In order to generate mutant reporter plasmids (mut-AC9), certain nucleotides in AC9 3′-UTR lacking miR-142-3p binding sites were mutated using RT-PCR. The constructed luciferase reporter plasmids were separately co-transfected with 50 nM miR-142-3p mimic or mimic NC into 293T cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were lysed using lysis buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) for 15 min at room temperature and luciferase activities of gene plasmid and phRL-TK were assayed at 48 h post-transfection using a Dual-Luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's instructions.
4
Bax 3'UTR Regulation by miR-29a-3p
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According to TargetScan 7.2 (http://www.targetscan.org/vert_72/ ), a conserved binding site of miR-29a-3p was found in the 3′UTR of Bax. The 3′UTR of Bax containing the predicted binding site of miR-29a-3p was cloned into the pmirGLO firefly luciferase reporter vector (Promega Corporation). The PCR procedures were as follows: A hot start step at 95°C for 10 min, 40 cycles at 95°C for 15 sec and 55°C for 45 sec, then a final step of 72°C for 30 sec.
For the luciferase reporter assay, cells were seeded at 5×104 cells/well in 24-well plates in 500 µl DMEM (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (HyClone; GE Healthcare Life Sciences), 100 U/ml penicillin (HyClone; GE Healthcare Life Sciences) and 100 µg/ml streptomycin (HyClone; GE Healthcare Life Sciences) for 18 h at 37°C. Then, the modified firefly luciferase vector (500 ng/µl) was mixed with Vigofect transfection reagent (Vigorous Biotechnology Beijing Co., Ltd.) according to the manufacturer's protocol. After transfection for 48 h, the Dual-luciferase Reporter Assay system (Promega Corporation) was applied to determine the changes in relative light units caused by the luciferase activity. Renilla luciferase activity was used to normalize the firefly luciferase activity.
For the luciferase reporter assay, cells were seeded at 5×104 cells/well in 24-well plates in 500 µl DMEM (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (HyClone; GE Healthcare Life Sciences), 100 U/ml penicillin (HyClone; GE Healthcare Life Sciences) and 100 µg/ml streptomycin (HyClone; GE Healthcare Life Sciences) for 18 h at 37°C. Then, the modified firefly luciferase vector (500 ng/µl) was mixed with Vigofect transfection reagent (Vigorous Biotechnology Beijing Co., Ltd.) according to the manufacturer's protocol. After transfection for 48 h, the Dual-luciferase Reporter Assay system (Promega Corporation) was applied to determine the changes in relative light units caused by the luciferase activity. Renilla luciferase activity was used to normalize the firefly luciferase activity.
5
Validating miR-429 Regulatory Interactions
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The binding sites between miR-429 and circ-PVT1, and between FOXK1 and miR-429 were predicted using ENCORI (http://starbase.sysu.edu.cn/index.php ). The wild-type (WT) or mutant (MUT) 3′-untranslated regions (UTRs) of circ-PVT1 and FOXK1 were constructed and cloned downstream of the pmirGLO firefly luciferase reporter vector (Promega Corporation), which were co-transfected with miR-429 mimic or mimic-NC into A549 cells using Lipofectamine 2000 reagent. Mutations in the 3′-UTRs were generated using a QuikChange Multi Site-Directed Mutagenesis kit (Agilent Technologies, Inc.). The relative luciferase activity was measured using a Dual Luciferase Reporter Assay kit (Promega Corporation) at 48 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.
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