The 3′-UTR of DAAM1 which contained the putative target site of miR-208a-5p was synthesized by Integrated Biotech Solutions Co., Ltd (Shanghai, China) and ligated into pGL3 construct (Promega, Madison, WI). The constructs pGL3-DAAM1-3′-UTR-WT or pGL3-DAAM1-3′-UTR-mutant (200 ng) and pRL-TK (80 ng, Promega) were co-transfected with 60 pmol miR-208a-5p mimic or miRNA mimic control by Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, Dual-Luciferase Reporter Assay System (Promega) was performed to measure luciferase activity.
Pgl3 construct
Manufactured by Promega
Sourced in China, United States
The PGL3 construct is a plasmid vector designed for gene expression studies. It contains a reporter gene that can be used to measure the activity of a promoter of interest. The PGL3 construct is commonly used in research laboratories to study gene regulation and transcriptional activity.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Prl tk, by Promega (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase assay, by Promega (1 mentions)
Psicheck2 construct, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Prl cmv plasmid, by Promega (1 mentions)
Pgl3 control vector, by Promega (1 mentions)
6 protocols using pgl3 construct
1
Validation of miR-208a-5p target site
2
Validating miR-342-3p Binding to SOX6 3'UTR
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Bioinformatics software TargetScan (http://www.targetscan.org/vert_72/ ) was used to predict the bindings sites of miR-342-3p in the 3′UTR of SOX6, and the results indicated the binding sites between the 3′UTR of SOX6 and miR-342-3p. Subsequently, to confirm the binding sites between SOX6 and miR-342-3p, dual-luciferase activity assay was performed. The 3′-UTR of SOX6, which contained the putative target site of miR-342-3p, was ligated into the pGL3 construct (Promega Corp.). The pGL3-SOX6-3′-UTR (wild-type) or pGL3-SOX6-3′-UTR (mutant) (200 ng) and pRL-TK (80 ng; Promega Corp.) constructs were co-transfected into the cells with 60 pmol miR-342-3p mimic or miRNA mimic control using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). Primers were synthesized by Genscript Nanjing Inc., and the sequences are presented in Table SI . At 24 h following transfection, the Dual Luciferase Reporter Assay System (Promega Corp.) was used to measure the luciferase activity and the luciferase activity was normalized to Renilla luciferase activity.
3
Luciferase Assay for miRNA Targets
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Fragments (540 bp) of VE‐cadherin and Notch4 3′UTR containing mutant or wild‐type miRNA‐responsive elements were cloned into the psiCheck2 construct (Promega) downstream of the Renilla luciferase ORF. Fragments (2600 bp) of circRNA7 promoter containing mutant or wild‐type miRNA‐responsive elements were cloned into the pGL3 construct (Promega) downstream of the Renilla luciferase ORF. SK cells were plated in 24‐well plates for 24 hours; then, the DNA was transfected into SK cells with Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Luciferase activity was assayed by dual‐luciferase assay (Promega), according to the manufacturer's protocol.
4
Validation of FOXM1-miR-149-5p interaction
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The sequences of wild-type (WT) or mutated (Mut) FOXM1 were purchased from Sangon (Shanghai, China) and then cloned into pGL3 construct (Promega Corporation, Madison, USA). MiR-149-5p mimics and corresponding reporter expression cassette were co-transfected into indicated cells by Lipofectamine 2000 as manual described. At 24 h post-transfection, relative firefly luciferase activity after normalized to renilla was detected by dual-luciferase reporter assay kit (Promega Corp.).
5
Validating miR-124-3p Targets via Luciferase Assay
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MiR-124-3p targets were validated by using pGL3 constructs (Promega, Madison, WI, USA). First of all, miR-124-3p target sequences were cloned into pGL3 using XhoI and NotI. Then the gene segments of mouse Caveolin-1 3′UTR were amplified from the genomic DNA using primers as indicated in Table 2 . DH-5α bacterial strains were co-transfected with 100 pM miR-124-3p mimics, 400ng pGL3-control or pGL3-Cav1- 3′UTR-WT or pGL3-Cav1-3′UTR-MUT, and 200ng pRL-TK vectors in 24-well plates. According to the manufacturer's instructions, the relative luciferase activities of firefly and Renilla were measured using the Dual-Luciferase Reporter Assay System (Promega). 1) 50 ul of Luciferase Assay Reagent II was added to detect the background value generated by the reagent. 2) 20 ul of PLB cracking liquid was added to detect the signal of firefly luciferase which was on behalf of the activity of Caveolin-1. 3) 50 μl Stop & Glo Reagent was used to measure the signal of Renilla luciferase which could be the normalization. 4) The relative activities of Caveolin-1 were the quotient of firefly/ Renilla luciferase activities, and three independent experiments were performed in triplicate.
6
TP63 3'UTR Luciferase Assay
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The full fragment of TP63 3'UTR containing the potential binding site of miR-140 was amplified using PCR and the binding site was mutated using a Quik-Change Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA, USA). Subsequently, wild-type and mutant TP63 3'UTR was inserted into a pGL3-control vector (Promega, Madison, WI) immediately downstream of the luciferase reporter gene to generate different luciferase constructs. The cells were plated into 96-well plates at a final density of 1×10 5 cells/well and cultured for 12 hours, followed by co-transfection with 0.5 mg pGL3 constructs, 0.07 mg pRL-CMV plasmids (Promega, Madison, WI), and a miR-140 mimic or inhibitor. The transfection was carried out using Lipofectamine 3000 (Invitrogen, CA, US). At 48 hours post-transfection, a Dual Luciferase Reporter Assay System (Promega, Madison, WI) was utilized to measure the activity of firefly and renilla luciferase. The expression of different constructs was calculated by normalizing the firefly signal to the activity of renilla luciferase. Each test was repeated three times.
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