The lentiviral vector pLVX-3×Flag-Puro was used to clone the cDNA of human CLCF1 to obtain the pLVX-CLCF1-3×Flag-Puro plasmid. The pmirGLO dual-luciferase reporter from Promega was used to clone the human CLCF1 3′-UTR to obtain the wild-type pmirGLO-CLCF1-3′-UTR reporter. In the mutant reporter pmirGLO-CLCF1-3′-UTR-mut, mutations of seven nucleotides in the CLCF1 3′-UTR that correspond to the miR-30a-5p seed sequences were performed. DNA sequencing was performed to confirm all constructs. The agomiR-30a-5p, miR-30a-5p mimics, CLCF1 siRNA and their respective control RNAs and anti-miR-30a-5p reagents were procured (RiboBio, China).
Pmirglo dual luciferase reporter
Manufactured by Promega
Sourced in United States
The PmirGLO Dual-Luciferase Reporter Assay is a laboratory equipment product that measures the activity of two different luciferase enzymes simultaneously. It is designed to provide a reliable and sensitive method for the evaluation of microRNA (miRNA) activity and target validation.
Automatically generated - may contain errors
Lab products found in correlation
Dual glo luciferase assay system, by Promega (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Mir 30a 5p mimic, by RiboBio (1 mentions)
Luciferase reporter assay reagent, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Glomax 20 20 system, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Phusion enzyme, by New England Biolabs (1 mentions)
In fusion cloning kit, by Takara Bio (1 mentions)
Synergy h4 microplate reader, by Agilent Technologies (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
11 protocols using pmirglo dual luciferase reporter
1
CLCF1 regulation by miR-30a-5p
2
miR-16 Regulation of TAB3 Expression
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TargetScanHuman 7.0 (http://www.targetscan.org/ ) and miRBase (http://www.mirbase.org/ ) microRNA databases were used to identify the target genes of miR-16. The primers of TAB3 3′-UTR and mutant TAB3 3′-UTR that may contain binding sites of miR-16 were designed and synthesized. The two kinds of target fragment were cloned into the pmirGLO dual Luciferase Reporter (Promega Corp., Madison, Wisconsin, USA) to construct the wild-type pmirGLO-TAB3 vector and mutant pmirGLO-TAB3 vector. Following that, the NP cells were transfected with the two types of vectors and miR-16 mimics or negative controls. After 24 h of transfection, the cells were collected. The luciferase was detected by the Luciferase reporter assay reagents (Promega Corp., Madison, Wisconsin, USA).
3
Luciferase Reporter Assay for miRNA Regulation
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The wildtype 3′UTR of MYCN containing the predicted target sites of the miRNAs were cloned from human genomic DNA. Mutant constructs with the seed target sequences for the corresponding miRNAs deleted were generated by site-directed mutagenesis. The 3′UTRs were cloned downstream of the firefly luciferase coding sequences into the pmirGLO dual-luciferase reporter (Promega) as previously described [14 (link)]. BE(2)-C cells were co-transfected with 0.8 ng/ul of the luciferase reporters and 25 nM of the mimics in 96-well plates. After 72 hours, Firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity to evaluate the regulation of firefly luciferase expression by the miRNAs.
4
Characterizing miR-2110 Binding Sites in 3'UTR
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DNA fragments (900bp) of the wild-type (WT) 3’UTR containing all three predicted target sites of miR-2110 were amplified from human genomic DNA. The amplified WT 3’UTR was inserted downstream of firefly luciferase coding sequences into pmirGLO dual-luciferase reporter (Promega), a vector that contains both firefly and Renilla luciferase cDNAs under the control of separate promoter/terminator systems, to generate the TSKU-WT reporter. Three mutant reporters with mutations of the seed sequences at each predicted target site was generated (TSKU-mut1, TSKU-mut2 and TSKU-mut3) using site-directed mutagenesis kit. A mutant reporter with all three predicted target sites mutated was also generated. BE(2)-C cells were co-transfected with the specified luciferase reporters (0.8 ng/μl) and oligos (miR-2110 mimic or control oligo, 25 nM). Luciferase activities were measured after 72 hours and the effect of miR-2110 was determined as previously described [14 (link)].
5
Luciferase Assay for miRNA Target Validation
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The segments of the wildtype 3'UTRs for CDK4 and STAT3 containing the predicted target sites of miR-506-3p and miR-124-3p were cloned from human genomic DNA. Mutant constructs were generated with the seed target site (GUGCCUU) replaced by CACGGUU. The 3'UTRs were cloned downstream of the firefly luciferase coding sequences into the pmirGLO dual-luciferase reporter (Promega), a vector containing both firefly and Renilla luciferase cDNAs under the control of separate promoter/terminator systems. The firefly luciferase was used as the primary reporter for miRNA regulation of the 3'UTR. The Renilla luciferase is an internal control for normalization. BE(2)-C cells were co-transfected with luciferase reporters (0.8 ng/ul) and miRNA mimics or control oligonucleotide (oligo) (25 nM). Luciferase activities were measured after 72 h using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity to evaluate the effect of the miRNAs.
6
Luciferase Assay for miR-9-3p Binding
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The binding site of SH2B3 3′ UTR (or MIR22HG) in miR-9-3p is predicted in starBase database and subcloned into the pmirGLO dual-luciferase reporter (Promega, USA) to construct wild-type or mutant pmirGLO-SH2B3-3′UTR (or pmirGLO-MIR22HG) vectors. The wild-type or mutant pmirGLO-SH2B3-3′ UTR (or pmirGLO-MIR22HG) vectors were cotransfected into HL-1 cells with miR-9-3p or NC mimics using Lipofectamine 2000 (Invitrogen). Luciferase activities were measured using dual-luciferase reporter assay system (Promega) after cotransfection for 48 h. The relative luciferase activity is expressed as the ratio of firefly/renilla luciferase activity. This assay was performed for 3 times (n = 3).
7
Luciferase Assay for miRNA-155-5p Target Validation
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Luciferase reporter gene assays were performed as previously described with some modifications.21 The online tool, TargetScanHuman,22 was used to predict possible miR‐155‐5p‐binding sites in SOCS1. Fragments of the wild‐type 3′ untranslated region (UTR) of SOCS1 containing the predicted target sites of miR‐155‐5p were cloned into the pmirGLO dual‐luciferase reporter (Promega Corporation,). Mutant constructs were generated with the target sites mutated, as specified in the “Results” section. Firefly luciferase was used as the primary reporter to investigate miRNA binding to the 3′‐UTR and subsequent regulation of gene expression. Renilla luciferase was used as an internal control for normalization. Human embryonic kidney 293T cells were seeded in 96‐well plates and co‐transfected with luciferase reporters (0.05 μg/well) and an miR‐155‐5p agomir or a SNC oligo (15 nM). Luciferase activities were measured 72 hours later using the Promega GloMax 20/20 system. Firefly luciferase activity was normalized to Renilla luciferase activity to assess the regulatory effect of miR‐155‐5p on its putative targets.
8
miR-192-5p Binding Site Validation
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The TRIP13 3′UTR sequence and the target-site mutant 3′UTR sequence were amplified and cloned into the pmirGLO DUAL-luciferase reporter (Promega). HCC cells were co-transfected with TRIP13 3′-UTR-wt or TRIP13 3′-UTR-mut, and miR-192-5p mimics, miR-control using the Lipofectamine 2000 reagent (Invitrogen, USA). Following cultivation for 48 h, the transfected cells were collected and detected for luciferase activity using a dual luciferase reporter assay kit (Promega, USA) based on the manufacturer’s instructions.
9
Validation of miR-506-3p target site in PLAGL2 3'UTR
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The pmiRGLO 3’UTR Luciferase reporter assay was used to validate the target site of miR-506-3p in the 3’UTR in the PLAGL2 mRNA. To generate the wildtype luciferase reporter (PLAGL2-WT) for the predicted miR-506-3p target site in the 3’UTR of PLAGL2, a fragment of the PLAGL2 3’UTR (3008 bp to 3671 bp) containing the predicted target site (UGCCUUA) of miR-506-3p was amplified from human genomic DNA by PCR using Phusion enzyme (New England Biolabs, Ipswich, MA, USA) and inserted downstream of firefly luciferase coding sequence (CDS) in the pmirGLO dual-luciferase reporter (Promega, Madison, WI, USA) using In-Fusion cloning kit (Takara Bio USA, Mountain View, CA, USA). Mutant reporter (PLAGL2-MU) with mutated seed sequence (CCUCAGA) at predicted target site was generated by site-directed mutagenesis using high fidelity DNA polymerase (Agilent Technologies, Santa Clara, CA, USA). BE(2)-C cells were co-transfected with pmiRGLO luciferase reporter and miR-506-3p for 2 days, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) in a BioTek Synergy H4 microplate reader.
10
Luciferase Reporter Assay for miRNA-31 Binding
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Regions containing the miRNA‐31‐bingding site of the human STK40 3′‐UTR was generated by PCR amplification and subcloned into the NheI/XbaI sites of the pmirGLO Dual‐Luciferase reporter plasmid (E1330; Promega). The mutations were generated with the predicted target site of STK40 3′‐UTR using the Q5® Site‐Directed Mutagenesis Kit (NEB, E0554), according to the manufacturer's instructions. The control luciferase plasmid, pmirGLO‐STK40‐3′UTR plasmid, or mutation plasmid was cotransfected into 293T cells with miRNA‐31 mimics using Lipofectamine 2000 Reagent (Invitrogen, Foster, USA). Luciferase activities were assayed by the Dual‐Luciferase Reporter Assay System (Promega) after 48 hours. Firefly luciferase (luc2) was used as the primary reporter to monitor miRNA regulation and Renilla luciferase (hRluc‐neo) acted as a control reporter for normalization and selection. Reduced firefly luciferase expression indicates the binding of introduced miRNAs to the cloned miRNA target sequence.
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