The entire 3’-UTR of SGK1 was amplified from human genomic DNA using PCR with the primer sequences: forward, 5’-ATAGCTAGCACCCTGTTAGGGCTTG and reverse, 5’-ATATCTCGAGCAACGGCTCTGACTG . The 3’-UTR was inserted into pmiRGLO Luciferase miRNA Target Expression Vector (Promega) and the sequence was confirmed by Sanger sequencing. Luciferase assays were conducted as previously reported following transfection with miRNA mimics or controls15 (link). β–Galactadase plasmid was co-transfected for normalization.
Pmirglo luciferase mirna target expression vector
Manufactured by Promega
Sourced in United States
The PmirGLO Luciferase miRNA Target Expression Vector is a plasmid-based system used to study microRNA (miRNA) target interactions. The vector contains a firefly luciferase reporter gene and multiple cloning sites to facilitate the insertion of miRNA target sequences. This system allows for the quantitative assessment of miRNA-mediated regulation of gene expression.
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6 protocols using pmirglo luciferase mirna target expression vector
1
Cloning and Luciferase Assay of SGK1 3'-UTR
2
Unveiling the miR-539-5p-LINC00339-TWIST1 Axis
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The potential miR-539-5p binding sites of LINC00339 were predicted with the help of bioinformatics databases (Starbase v2.0). The putative miR-539-5p target binding sequence in LINC00339 and its mutant of the binding sites were synthesized and cloned into the pmirGLO luciferase miRNA Target Expression vector (Promega, Madison, WI, USA).
The target genes of miR-539-5p were predicted using the bioinformatics databases miRanda (http://www.MiRanda.org ). To examine whether miR-539-5p targets TWIST1 directly, we constructed wild-type TWIST1 reporter plasmid (TWIST1-Wt) and mutated-type TWIST1 reporter plasmid (TWIST1-Mut) with pmirGLO-promoter vector (GenePharma, Shanghai, China), respectively.
HEK293T cells were seeded in 96-well plates for 1 day, and cells at 60%–80% confluence were co-transfected with the pmirGLO vector constructed with either wild-type or mutation type and miR-539-5p overexpression or miRNA NC plasmids using Lipofectamine 3000 Reagents according to the manufacturer’s instructions. The luciferase activity was measured 2 days after transfection using a dual luciferase assay kit (Promega, Madison, WI, USA), and the relative firefly luciferase activity was expressed as the ratio of firefly luciferase activity to renilla luciferase activity. All transfections were performed in triplicate.
The target genes of miR-539-5p were predicted using the bioinformatics databases miRanda (
HEK293T cells were seeded in 96-well plates for 1 day, and cells at 60%–80% confluence were co-transfected with the pmirGLO vector constructed with either wild-type or mutation type and miR-539-5p overexpression or miRNA NC plasmids using Lipofectamine 3000 Reagents according to the manufacturer’s instructions. The luciferase activity was measured 2 days after transfection using a dual luciferase assay kit (Promega, Madison, WI, USA), and the relative firefly luciferase activity was expressed as the ratio of firefly luciferase activity to renilla luciferase activity. All transfections were performed in triplicate.
3
Validation of CXCR4 miRNA Binding Site
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The predicted binding site in the 3'-UTR of CXCR4 was elaborated by PCR, and inserted into the pmir GLO Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA), which was known as CXCR4 wild type (CXCR4-WT). The predicted binding sites were substituted for forming the negative control: CXCR4 mutated type (CXCR4-MUT). The vectors were transfected with miR-139 mimic or the mimic NCs with Lipofectamine 3000 reagent. After 48 h of transfection, the luciferase activities were detected with the Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
4
Validation of ABCA1 3'-UTR Interactions
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To analyze ABCA1 3′-UTRs containing the miR-33a-5p, 33b-5p, and 148a-3p binding sites, we constructed a pmirGLO luciferase miRNA target expression vector (Promega, Madison, WI, USA) and verified the results of sequencing (Bioneer, Daejeon, Republic of Korea). Human umbilical vein endothelial cells (HUVECs) were seeded on 24-well plates and transfected with each miRNA mimic or negative control (NC) of 20nM using Lipofectamine 2000 (Invitrogen) for 24 h. The relative luciferase activities were measured by luminescence (Tecan, Switzerland).
5
Validating miR-542-3p Binding Sites
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The putative binding sites of miR-542-3p for SNHG8 as well as AP3D1 were predicted by starBase (http://starbase.sysu.edu.cn ). MiR-542-3p cDNA was cloned into the pmirGLO Luciferase miRNA Target Expression Vector (Promega) to form the pmirGLO-miR-542-3p-WT reporter vector. The mutant miR-542-3p was specifically synthesized and inserted into the pmirGLO vector to construct the pmirGLO-miR-542-3p-Mut reporter vector. sh-NC and sh-SNHG8 were transfected with pmirGLO-miR-542-3p-WT or pmirGLO-miR-542-3p-Mut into THP-1-derived macrophages. For validating the interaction of miR-542-3p and AP3D1 3ʹUTR, the wild-type and mutant AP3D1 3ʹ-UTR fragments were cloned into the pmirGLO vector to form pmirGLO-AP3D1-WT and pmirGLO-AP3D1-Mut, respectively. The miR-542-3p mimics, NC mimics, or miR-542-3p mimics + SNHG8 was cotransfected with pmirGLO-AP3D1-WT and pmirGLO-AP3D1-Mut into THP-1-derived macrophages using Lipofectamine 3000 (Invitrogen). After transfection for 48 h, the luciferase assay was performed with the employment of the Luciferase Reporter Assay System (Promega). Relative firefly luciferase activity was normalized to Renilla luciferase activity.
6
Luciferase Reporter Assay for miR-4656 Binding
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The regions of Linc003339 or 3′UTR locus of CSNK2B, containing miR-4656-binding sites was amplified and cloned into pmirGLO luciferase miRNA Target Expression vector (Promega, Madison, WI, USA). The mutant reporter plasmids were constructed as described previously (27 (link)). The wide type plasmids (WT) or mutant type plasmids (Mut) were co-transfected with miR-4656 or NC into breast cancer cells. Firefly and Renilla luciferase activities were measured with the dual-luciferase reporter assay kit (Promega, Madison, WI, USA) 2 days after transfection. The relative luciferase activity was measured as the ratio of firefly luciferase activity to renilla luciferase activity. All experiments were performed in triplicate.
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