The full‐length human Src 3′‐UTR and the mutant Src 3′‐UTR were cloned into the p‐MIR‐reporter vector (Ambion, Austin, TX), respectively. TE‐1 cells were co‐transfected with the p‐MIR‐reporter vector, β‐galactosidase (β‐gal) expression vector (Ambion), and 10 pmol of miR‐1 mimic or scrambled negative control RNA. Relative luminescences were collected 24 h posttransfection using the luciferase assay kit (Promega, WI).
Pmir reporter vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PMIR-REPORTER vector is a plasmid-based tool used for the expression and detection of microRNA (miRNA) activity in cells. It contains a reporter gene that is regulated by a miRNA-responsive element, allowing for the monitoring of miRNA expression levels. This vector provides a standardized platform for miRNA functional studies.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (13 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Dual luciferase assay kit, by Promega (4 mentions)
Synthetic oligonucleotides, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Pgl4.74 vector, by Thermo Fisher Scientific (2 mentions)
Luciferase assay kit, by Promega (2 mentions)
P mir reporter vectors, by Thermo Fisher Scientific (2 mentions)
Fugene hd transfection agent, by Promega (1 mentions)
Dual luciferase assay, by Promega (1 mentions)
Prl cmv, by Promega (1 mentions)
Cel mir 67, by Horizon Discovery (1 mentions)
Dual glo luciferase reporter assay system, by Promega (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Prl sv40, by Promega (1 mentions)
Dual glo luciferase assay, by Promega (1 mentions)
Quickchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Purelink hipure plasmid filter purification kit, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
33 protocols using pmir reporter vector
1
Src 3'UTR Luciferase Assay
2
Luciferase Reporting of miRNA-SREBF1 Interactions
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The 3′ UTR segments of SREBF1, predicted to interact with miRNAs, were amplified by PCR from human genomic DNA and inserted into the pMIR-reporter vector (Applied Biosystems), using the MluI and HindIII sites immediately downstream from the luciferase stop codon.
For the transfection and luciferase reporter experiments, the human embryonic kidney cell line 293T was grown in 10% FBS in DMEM medium at 37 °C in a humidified atmosphere with 5% CO2. The cells were transfected in 96-well plates using Lipofectamine® 2000 (Life Technologies) according to the manufacturer’s protocol. 0.2 μg of the firefly luciferase report vector and 0.01 μg of the control vector containing Renilla luciferase, pRL-CMV (Promega), or 100 nM miRNA oligonucleotides (Shanghai Gene Pharma Co. Ltd) were used for transfection in each well. Firefly and Renilla luciferase activities were determined consecutively using dual-luciferase assays (Promega) 48 h after transfection.
The miRNA mimics (ago-miR-185-5p), inhibitors (antago-miR-185-5p), and negative controls of miR-185-5p were purchased from RiboBio (RiboBio, Guangzhou, China). The cells were transfected with a 50 nM solution of the mimic, inhibitor, and negative controls. The Fu GENE HD transfection agent (Promega, USA) was used according to the manufacturer’s instructions.
For the transfection and luciferase reporter experiments, the human embryonic kidney cell line 293T was grown in 10% FBS in DMEM medium at 37 °C in a humidified atmosphere with 5% CO2. The cells were transfected in 96-well plates using Lipofectamine® 2000 (Life Technologies) according to the manufacturer’s protocol. 0.2 μg of the firefly luciferase report vector and 0.01 μg of the control vector containing Renilla luciferase, pRL-CMV (Promega), or 100 nM miRNA oligonucleotides (Shanghai Gene Pharma Co. Ltd) were used for transfection in each well. Firefly and Renilla luciferase activities were determined consecutively using dual-luciferase assays (Promega) 48 h after transfection.
The miRNA mimics (ago-miR-185-5p), inhibitors (antago-miR-185-5p), and negative controls of miR-185-5p were purchased from RiboBio (RiboBio, Guangzhou, China). The cells were transfected with a 50 nM solution of the mimic, inhibitor, and negative controls. The Fu GENE HD transfection agent (Promega, USA) was used according to the manufacturer’s instructions.
3
ARID4A 3'UTR miR-376c Binding Assay
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The 3′-UTR sequence of the ARID4A gene which contains predicted miR-376c binding site was cloned into the pMIR-REPORTER vector (Applied Biosystems) to generate the reporter construct designated pMIR-ARID4A. The plasmid pRL-TL, which expresses the renilla luciferase gene, was used as a transfection control. After co-transfecting of miR-376c mimic with pMIR-REPORTER (vector alone; VA) or pMIR-ARID4A, as well as pRL-TL for 24 hours, the lysate were collected and assayed in accordance with the protocol of Dual-Luciferase® reporter assay system (Promega, WI, USA). Firefly luciferase activity was normalized against renilla luciferase activity to indicate the reporter activity.
4
miRNA and siRNA Dual Reporter Assay
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All the reporters were generated by inserting synthetic oligonucleotide DNA linkers of MRE sequences or their mutants into the pMIR-REPORTER vector (Applied Biosystems) downstream of luciferase gene through Hind III and Spe I sites. Native RNA duplexes for miR-146b and siEGFP (siRNA against EGFP) were custom-synthesized by Integrated DNA Technology. miRIDIAN miR-146b mimic and a negative control (cel-miR-67, which has no sequence identity with miRNAs in human, mouse and rat) were purchased from Dharmacon. Locked nucleic acid (LNA) anti-sense oligonucleotides were purchased from Exiqon, Inc.
5
miR-15b-5p Binding Assay on HPSE2 3'UTR
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The wild-type (WT) and mutant (Mut) binding sequences of miR-15b-5p on the 3′ UTR of HPSE2 were synthesized (GenePharma, Shanghai, China) and cloned into the pMIR-reporter vector (Invitrogen). Cells (MCF-7 and 293T) were seeded into 96-well-plates at a density of 1 × 104 cells per well. When the cells spread to nearly 80–90% of the bottom of 96-well-plates, they were transfected with luciferase reporters, either WT or Mut HPSE2 3′ UTR, with or without miR-15b-5p mimics or NC by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Forty-eight hours later, the quantitative analysis of firefly and renilla luciferase activity via the Dual-Glo Luciferase Reporter Assay system (Promega, Madison, WI, USA) on a multifunctional enzyme-linked immunosorbent assay microplate reader (Turner BioSystems, Sunnyvale, CA, USA) per the manufacturer's operation manual. For data analysis, firefly luciferase activity was normalized to the corresponding renilla luciferase activity. All experiments were implemented in triplicate and repeated three times.
6
Luciferase Assay for miR-143-3p Binding
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The human LINC02470 miR-143-3p binding fragment and the SMAD3 3′UTR containing the miR-143-3p binding site were amplified via PCR from human genomic DNA using the primers listed in Table S1 and were inserted between the SpeI/HindIII sites in a pMIR-reporter vector (Invitrogen, Waltham, MA, USA). All constructs were verified by autosequencing.
A total of 5000 T24 cells were seeded into a 24-well plate 16 h before transfection. For the miR-143-3p binding assay, cells were co-transfected with 120 pmol of miR-143-3p mimic or miR-NC control and with 1 µg of pMIR-reporter empty vector or pMIR-reporter carrying the wildtype or mutant LINC02470 miR-143-3p binding fragment or SMAD3-3’UTR using Lipofectamine 2000 (Invitrogen). In addition, cells were co-transfected with 500 ng of β-gal control vector for each combination as an internal control. Luciferase assays were performed with a Dual-Light Luciferase and β-Galactosidase Reporter Gene Assay System (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Luminescence was detected using a SpectraMax i3x (Molecular Devices LLC, San Jose, CA, USA), and six replicates were prepared for each condition.
A total of 5000 T24 cells were seeded into a 24-well plate 16 h before transfection. For the miR-143-3p binding assay, cells were co-transfected with 120 pmol of miR-143-3p mimic or miR-NC control and with 1 µg of pMIR-reporter empty vector or pMIR-reporter carrying the wildtype or mutant LINC02470 miR-143-3p binding fragment or SMAD3-3’UTR using Lipofectamine 2000 (Invitrogen). In addition, cells were co-transfected with 500 ng of β-gal control vector for each combination as an internal control. Luciferase assays were performed with a Dual-Light Luciferase and β-Galactosidase Reporter Gene Assay System (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Luminescence was detected using a SpectraMax i3x (Molecular Devices LLC, San Jose, CA, USA), and six replicates were prepared for each condition.
7
Irf2 mRNA Luciferase Assay
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The putative miR-221/222 complementary site in the 3′-UTR of Irf2 mRNA or its mutant sequence was cloned into the p-MiR-reporter vector (Ambion, USA). For luciferase reporter assay, BV2 cells were seeded in 96-well plates and then cotransfected with p-MiR-reporter vectors with miR-221, miR-222 mimics, or control mimics using Lipofectamine 2000 (Invitrogen, USA). pRL-SV40 (Promega, WI, USA) was used as the control vector to balance transfection efficiency. After transfection for 48 h, the relative luciferase activity was detected using a dual-luciferase reporter assay system (Promega, USA).
8
Dual-Luciferase Assay for miR-199a Binding
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3′-UTR region of K-RAS containing software predicted miR-199a-matching seed sites (WT) and corresponding mutant sites (mut) were amplified by High fidelity PCR, and inserted into pMIR-REPORTER vector (Ambion, USA). Dual-luciferase activities were analyzed in U87 cells by the Reporter Assay (Promega, USA).
9
Validation of EDDM3A 3'UTR-miRNA Interaction
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The wild-type and mutant 3′UTR sequences of EDDM3A were amplified from the full-length cDNA and subcloned into the pMIR-Reporter vector (Ambion) to obtain 3′-UTR-luciferase reporter plasmid. Mutation of the miRNA seed binding sites was performed using the QuickChange site-directed mutagenesis kit (Agilent). The plasmids were respectively named as EDDM3A 3′UTR-WT and EDDM3A 3′UTR-mut. Each vector along with miR-618 or scrambled control were cotransfected into GC cells using Lipofectamine 3000 reagent (Invitrogen) following the manufacturer’s instructions. Twenty-four hours later, the luciferase activity was measured by Dual-Glo luciferase assay (Promega).
10
Dissecting miR-6778-5p and SHMT1 Interaction
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