The wild-type (WT) sequence of lncRNA-KCNQ1OT1 containing the miR-141-5p binding sites (KCNQ1OT1-WT) and the mutant sequence (KCNQ1OT1-MUT) were cloned into pMIR vectors (Promega, USA), respectively. Primary osteoblasts and HEK293 cells were cotransfected with miR-141-5p or miR-NC and KCNQ1OT1-WT or KCNQ1OT1-MUT. The luciferase activity was detected using Dual-Luciferase Reporter Assay System (Promega, USA) in the dark.
Pmir vector
Manufactured by Promega
Sourced in United States
The PMIR vector is a plasmid designed for the expression of microRNA (miRNA) in mammalian cells. It contains the necessary elements for the transcription and processing of miRNA, including a human cytomegalovirus (CMV) promoter and the flanking sequences required for miRNA processing.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (8 mentions)
Dual luciferase reporter system, by Promega (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase assay, by Promega (1 mentions)
Prl tk vector, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Elecsys 2010, by Roche (1 mentions)
Geneart gene synthesis, by Thermo Fisher Scientific (1 mentions)
Rapid dna extraction and detection kit, by Tiangen Biotech (1 mentions)
Lumat lb 9507 luminometer, by Berthold Technologies (1 mentions)
Dual luciferase reporter assay system kit, by Promega (1 mentions)
Lipo2000, by Thermo Fisher Scientific (1 mentions)
Glomax 96 microplate luminometer, by Promega (1 mentions)
Veritas microplate luminometer, by Promega (1 mentions)
17 protocols using pmir vector
1
Elucidating lncRNA-KCNQ1OT1 Regulation
2
Validation of miR-505 Binding to sUmod
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GenePharma (Shanghai, China) was also responsible for preparing mutant (MUT) and wild-type (WT) 3′-UTR in sUmod. Thereafter, MUT or WT 3′-UTR that contained mutations of sUmod binding sites was cloned in downstream luciferase within the empty pMIR vectors (Promega, USA) for producing the recombinant constructs (MUT- or WT-sUmod, separately). Afterwards, by adopting lipofectamine 2000, these constructs were cotransfected into VSMCs with miR-control or miR-505 mimics. In addition, cells were cotransfected with pRL-TK vector (Promega, USA) as well as Renilla luciferase gene for control. At 48 h later, dual luciferase assay (Promega, USA) was utilized to measure Renilla and firefly luciferase activities in line with specific protocols.
3
Luciferase Assay for SMAD2 3'UTR
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The recombinant luciferase plasmids using pMIR vectors (Promega, Madison, WI, United States) harboring SMAD2 3' untranslated region (3'UTR) and SMAD2-mut 3'UTR with mutant binding regions for miR-423-5p were constructed. The above plasmids were transfected into human aortic VSMCs, which were divided into five groups: pMIR vector, pMIR-SMAD2-plasmid + NC, pMIR-SMAD2-plasmid + miR-423-5p mimics, pMIR-SMAD2-mut-plasmid + NC, and pMIR-SMAD2-mut-plasmid + miR-423-5p mimics. Relative luciferase activity was examined using Dual-Luciferase Reporter Assay System (Promega) as the ratio of firefly/Renilla luciferase activity. The enzymatic activities of firefly and control Renilla luciferases were sequentially measured in every single sample using Dual-Luciferase Reporter Assay System (Promega) on addition of Stop & Glo® Reagent to the reaction. Relative luciferase activity was examined as the ratio of firefly/Renilla luciferase activity.
4
Dual Luciferase Reporter Assay for PLCE1 3'UTR
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ESCC cells were maintained in DMEM; cells at 70–90% confluence in 96-well plates were allowed to settle for 16 h, and then co-transfected with 200 ng of pMIR vectors (Promega), which harbored PLCE1 3′-UTR wild-type or mutant constructs and 10 ng of the Renilla luciferase vector with Lipofectamine® 2000 Reagent (Invitrogen) according to the recommendation of the manufacturer. Luciferase and Renilla signals were measured at 48 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer. Three independent experiments were performed and the data were presented as the mean ± SD.
5
Luciferase Assay for lncRNA-KCNQ1OT1 Binding
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The wild-type (WT) sequence of lncRNA-KCNQ1OT1 containing the miR-141-5p binding sites (KCNQ1OT1-WT) and the mutant sequence (KCNQ1OT1-MUT) were cloned into pMIR vectors (Promega, USA) respectively. Primary osteoblasts and HEK293 cells were co-transfected with miR-141-5p or miR-NC and KCNQ1OT1-WT or KCNQ1OT1-MUT and kept for 24 h. The luciferase activity was detected using Dual-Luciferase Reporter Assay System (Promega, USA) in the dark.
6
Luciferase Assay for lncRNA-KCNQ1OT1 Binding
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The wild-type (WT) sequence of lncRNA-KCNQ1OT1 containing the miR-141-5p binding sites (KCNQ1OT1-WT) and the mutant sequence (KCNQ1OT1-MUT) were cloned into pMIR vectors (Promega, USA) respectively. Primary osteoblasts and HEK293 cells were co-transfected with miR-141-5p or miR-NC and KCNQ1OT1-WT or KCNQ1OT1-MUT and kept for 24 h. The luciferase activity was detected using Dual-Luciferase Reporter Assay System (Promega, USA) in the dark.
7
Luciferase Assay for miR-129 Targeting
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The wild-type human 3′ UTR of FBW7, which contains the predicted binding site for miR-129, was cloned into the pMIR vector (Promega, WI, USA). The mutant FBW7 3′ UTR was generated from wild-type FBW7 3′ UTR by site-directed mutagenesis. 2 × 105 Caco-2 cells were transfected with miR-129 mimics or miR-129 inhibitor or corresponding negative controls and wild-type or mutant 3′ UTR of FBW7 dual luciferase reporter vector using Lipofectamine 2000. After 48 h transfection, luciferase activities were quantified using a dual luciferase reporter system (Promega) on a luminometer (Elecsys 2010; Roche Diagnostics, Basel, Switzerland).
8
Targeted Mutagenesis of SMAD4 3'UTR
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The 3′UTR of the SMAD4 gene was obtained from GeneArt Gene Synthesis (Invitrogen, Thermo Fisher Scientific) by cloning 900bp of SMAD4 3′UTR into pMir-vector (Promega) giving rise to the pMir-3′UTRSMAD4 construct.
Site-direct mutagenesis into the miR-205, miR146a, and miR-1260b binding sites of the SMAD4 gene 3′UTR were introduced using GeneArt Site-Directed Mutagenesis PLUS System kit (Thermo Fisher Scientific) according to manufacturer's instructions. The primers used were:
FP 5′CTTCACCTGTTATGTAcctgccAATCATTCCAGTGC3′
RP 5′GCACTGGAATGATTggcaggTACATAACAGGTGAAG3′
FP 5′GCTGATTTTAAAGGCAGAGAAccgtcgAAAGTTAATTCACC3′
RP 5′GGTGAATTAACTTTcgacggTTCTCTGCCTTTAAAATCAGC3′
FP 5′GTTATTCCTAGTGacccgtTGTTGATGAAGTATACTTTTCCCC3′
RP 5′GGGGAAAAGTATACTTCATCAACAacgggtCACTAGGAATAAC3
Site-direct mutagenesis into the miR-205, miR146a, and miR-1260b binding sites of the SMAD4 gene 3′UTR were introduced using GeneArt Site-Directed Mutagenesis PLUS System kit (Thermo Fisher Scientific) according to manufacturer's instructions. The primers used were:
FP 5′CTTCACCTGTTATGTAcctgccAATCATTCCAGTGC3′
RP 5′GCACTGGAATGATTggcaggTACATAACAGGTGAAG3′
FP 5′GCTGATTTTAAAGGCAGAGAAccgtcgAAAGTTAATTCACC3′
RP 5′GGTGAATTAACTTTcgacggTTCTCTGCCTTTAAAATCAGC3′
FP 5′GTTATTCCTAGTGacccgtTGTTGATGAAGTATACTTTTCCCC3′
RP 5′GGGGAAAAGTATACTTCATCAACAacgggtCACTAGGAATAAC3
9
miR-144-3p Regulates ANGPTL3 Expression
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The potential interaction between the 3′-untranslated region (3′-UTR) of ANGPTL3 and miR-144-3p was predicted using the TargetScan platform (https://www.targetscan.org/vert_80/ ). This was experimentally verified through luciferase reporter assay. The assay used synthetic versions of both the wild-type (WT) and mutant-type (MUT) seed regions of ANGPTL3, inserted into the pMIR vector (by Promega Corporation, Madison, WI, USA). To assess the impact of miR-144-3p on ANGPTL3, HK-2 cells underwent co-transfection with either a miR-144-3p inhibitor or a miR-NC, in combination with either pMIR-ANGPTL3-WT or pMIR-ANGPTL3-MUT, employing Lipofectamine 2000 (Invitrogen, USA) at a temperature of 37 °C. After incubating for 48 h, the cell lysates were collected for analysis. The measurement of relative luciferase activity (firefly versus renilla luciferase fluorescence ratio) was conducted using the Dual-Luciferase Reporter Assay System from Promega Corporation, adhering to the manufacturer's guidelines.
10
Validating miR-324-5p Binding Sites
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Wild and mutant sequences of ANRIL and Ran containing predicted miR-324-5p binding sites were amplified and inserted into the pMIR vector (Promega) using PCR. Then, vectors and miR-324-5p mimics were co-transfected into SKOV3 cells using Lipofectamine 3000. Forty-eight hours later, the luciferase activities were measured by a dual-luciferase reporter system (Promega) based on the manufacturer’s protocols and analyzed by normalizing Renilla activities.
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