The miR-98 mimics, inhibitor and corresponding negative control (NC) were obtained from Shanghai GenePharma Co., Ltd. The potential binding sequence between IGF1R and miR-98 was investigated using TargetScan (Version 7.1, http://www.targetscan.org ) and miRanda (Version 3.3a, http://www.microrna.org/microrna/home.do/ ). The wild-type (wt) and mutant (mut) human IGF1R 3′-untranslated regions (UTRs) containing the putative binding site of miR-98 were constructed, respectively and were subsequently cloned into a pMIR-REPORT luciferase reporter vector (Ambion; Thermo Fisher Scientific, Inc.). Site-directed mutagenesis of the miR-98 target-site in the IGF1R 3′-UTR was performed using a QuikChange kit (Qiagen, Inc.). For the luciferase assay, the Y79 cells were cultured in 96-well plates and co-transfected with 400 ng of pMIR-IGF1R-wt-3′-UTR or pMIR-IGF1R-mut-3′-UTR, and 50 ng miR-98 mimic, inhibitor or corresponding NC using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h following transfection, the relative firefly luciferase activity was normalized with that of Renilla luciferase as measured using a Dual-Light luminescent reporter gene assay (Applied Biosystems; Thermo Fisher Scientific, Inc.).
Pmir report luciferase reporter vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PMIR-REPORT luciferase reporter vector is a plasmid designed for the expression and detection of luciferase activity as a reporter gene. It contains the firefly luciferase coding sequence under the control of a promoter, allowing for the quantification of gene expression levels in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (11 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (10 mentions)
Luciferase assay kit, by Promega (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Quikchange kit, by Qiagen (3 mentions)
Dual light luminescent reporter gene assay, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (3 mentions)
Luciferase reporter assay system, by Promega (3 mentions)
Dual luciferase assay system, by Promega (3 mentions)
Site directed mutagenesis kit, by Takara Bio (2 mentions)
Dual luciferase assay, by Promega (2 mentions)
Prl tk, by Promega (2 mentions)
Lysis buffer, by Promega (2 mentions)
Veritas microplate luminometer, by Promega (2 mentions)
Luciferase assay system, by Promega (1 mentions)
Transmessenger transfection reagent, by Qiagen (1 mentions)
Prl tk renilla luciferase vector, by Promega (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Phusion high fidelity pcr kit, by Thermo Fisher Scientific (1 mentions)
Prl tk vector, by Promega (1 mentions)
33 protocols using pmir report luciferase reporter vector
1
Investigating miR-98 Regulation of IGF1R
2
Luciferase Assay for miR-17-5p Targeting HOXB13
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The 3′-UTR fragment of HOXB13 mRNA containing miR-17-5p-targeting sequence was cloned into a pMIR-REPORT luciferase reporter vector (Ambion; Thermo Fisher Scientific, Inc.) to create wild-type (WT) and mutated type (MT) pMIR-luc-HOXB13-3′-UTR vectors. VSMCs (1×104/well) were cultured in 24-well plates. When at 60–70% confluence, cells were co-transfected with the vectors (100 nM), together with 50 nM miR-175p mimics, 50 nM antagomiR-17-5p or 50 nM negative control oligonucleotides using Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.) at 37°C. A luciferase reporter vector without the miR-17-5p targeting sequence was transfected in parallel. After transfection for 48 h, luciferase activities were measured using the Dual-luciferase Reporter Assay system (Promega Corporation) and normalized to Renilla according to the manufacturer's instructions. The relative luciferase activity in cells was expressed as a percentage of the luciferase activity over that of cells transfected with the vector without miR-17-5p targeting sequence.
3
Validating miR-140-5p Targeting of c-Met
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The miR-140-5p mimics/inhibitor and corresponding NC were obtained from GenePharma (Shanghai, China). The potential binding site between c-Met and miR-140-5p was searched using TargetScan (Figure 3 A). The human c-Met 3′-UTR with wild-type (wt) and mutant (mut) containing the putative binding site of miR-140-5p were constructed (Figure 3 B) and then were cloned into a pMIR-REPORT luciferase reporter vector (Ambion, U.S.A.). Site-directed mutagenesis of the miR-140-5p target-site in the c-Met 3′-UTR was performed using a QuikChange Kit (Qiagen). For the luciferase assay, the Y79 cells were cultured in 96-well plates and co-transfected with 400 ng of either pMIR-c-Met-3′-UTR or pMIR-c-Met-mut-3′-UTR, and 50 ng of miR-98 mimic/inhibitor or corresponding NC using Lipofectamine 2000 reagent (Invitrogen). Forty-eight hours after transfection, the relative firefly luciferase activity normalized with Renilla luciferase was measured using the Dual-Light luminescent reporter gene assay (Applied Biosystems).
4
Elf3 3'UTR Luciferase Assay
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The 3′UTR of Elf3 with the wild‐type (WT) or mutant type (MUT) binding sites of miR‐206 were amplified and inserted into pMIR‐REPORT luciferase reporter vector (Ambion), defined as Elf3‐WT and Elf3‐MUT. The 293T cells were co‐transfected with 200 ng luciferase reporter vectors, 25 ng pRL‐TK (expressing Renilla luciferase as the internal control) and 20 μM miR‐206 mimic or miR‐206 mimic NC by Lipofectamine 2000 reagent. The luciferase report gene detection system (Promega Corporation) was used to analyse the luciferase activity after 24‐hours transfection.
5
Luciferase Assay for miR-203a-Smad3 Interaction
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The miR-203a mimics/inhibitor and corresponding NC were purchased from GenePharma (Shanghai, China). The potential binding site between Smad3 and miR-203a was searched using TargetScan (Figure 4 A) (http://www.targetscan.org ). The Smad3 3ʹ-UTR with wild-type (wt) and mutant (mut) containing the putative binding site of miR-203a were constructed (Figure 4 B) and subsequently cloned into a pMIR-REPORT luciferase reporter vector (Ambion, U.S.A.). The mutated plasmid, pMIR-Smad3-mut-3ʹ-UTR was generated by a QuikChange Kit (Qiagen). The chondrocytes were co-transfected with 400 ng of the reporter construct (pMIR-Smad3-3ʹ-UTR or pMIR-Smad3-3ʹ-UTR) and 50 ng miR-203a mimic/inhibitor or corresponding NC using Lipofectamine 2000 reagent (Invitrogen). Forty-eight hours after transfection, the relative firefly luciferase activity normalized with Renilla luciferase was measured using the Dual-Light luminescent reporter gene assay (Applied Biosystems).
6
Validating miR-155 Binding to CBL 3'UTR
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A luciferase reporter assay was employed to detect whether miR-155 bound to the 3′UTR of CBL directly. As predicted by TargetScan (http://www.targetscan.org/ ), fragments of CBL mRNA containing wild type (WT) or mutant (MUT) miR-155 binding sites or lacking the miR-155 binding site (DEL) were respectively cloned into the pMIR-REPORT luciferase reporter vector (Ambion, San Diego, CA, USA). HCT-116 cells were seeded into 24-well plates and 0.2 µg luciferase reporter plasmid WT, MUT or DEL, together with 0.2 µg β-galactosidase control plasmid (Ambion) plus either 20 pmol miR-155 mimics or negative control of mimics were co-transfected into HCT-116 cells using Lipofectamine 2000 reagent. At 48 h after transfection, luciferase activity was measured using a Luciferase Assay System (Promega Corp.) according to the manufacturer's protocols.
7
Luciferase Assay for Validating miR-20b Targets
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The target gene was predicted by TargetScan (http://www.targetscan.org/ ). A 450-bp fragment of the 3′-untranslated region (3′-UTR) of HIF-1α mRNA containing the target sequence (GCACTTT) of miR-20b was amplified by RT-PCR (HIF-1α, sense 5′-CTCTGAGCTCTATCTGGAAGGTATGTG-3′, antisense 5′-CCTCAAGCTTCAGTTAGTGTTAGACCC-3′). The fragment was designated HIF-1α 3′-UTR and inserted into the pMIR-REPORT™ luciferase reporter vector (SacI and HindIII restriction enzyme sites; Ambion). Another expressing vector was also constructed by the insertion of a mutated HIF-1α 3′-UTR in which the target sequence of miR-20b was mutated into GCAATTT using the QuikChangeH Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA) (22 (link)). The recombinant reporter vectors with normal and or mutated HIF-1α 3′-UTR were cotransfected with miR-20b mimic, miR-20b mimic control, miR-20b inhibitor, or miR-20b inhibitor control into MG63 using TransMessenger™ Transfection Reagent (Qiagen, Germany). The luciferase assay was performed according to the manufacturer’s instructions. The relative luciferase activities were normalized to that of the control cells.
8
Evaluating miR-519d Regulation of MCL-1 3'UTR
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To evaluate the function of miR-519d, the 3′ UTR of MCL-1 was amplified and inserted downstream of the luciferase reporter gene in the luciferase reporter pMIR-REPORT luciferase reporter vector (Ambion, Carlsbad, CA, USA). The mutant 3′UTR of MCL-1 was amplified using wild-type MCL-1 3 'UTR as the template, and the mutant plasmid was created by Site-Directed Mutagenesis Kit (TaKaRa). For luciferase reporter assays, the cells were co-transfected with miR-519d mimics and wild-type or mutant MCL-1 3′UTR, along with Renilla luciferase pRL-TK vector (Promega, USA) as an internal control. Following transfection for 48 h, the cells were collected and lysed using RIPA buffer (Cell Signaling Technology, USA). Luciferase activity was then measured by using Dual Luciferase Assay System (Promega) according to the manufacturer's instructions.
9
Luciferase Reporter Assay for miR-21-5p Binding
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The fragments of MEG3 and 3′-untranslated region (UTR) of SOX7 containing the predicted wild-type (WT) binding sites of miR-21-5p or mutated miR-21-5p binding sites (MUT) were amplified by PCR and inserted into a pMIR-REPORT luciferase reporter vector (Ambion, Austin, TX, USA), named as WT-MEG3, MUT-MEG3, WT-SOX7-3′UTR, and MUT-SOX7-3′UTR. When the luciferase reporter assay is carried out, cells were cotransfected with 200 ng constructed luciferase reporter vectors, 25 ng pRL-TK (expressing renilla luciferase as the internal control) and 20 μM miR-21-5p or miR-NC using Lipofectamine 2000. A luciferase reporter assay system (Promega Corporation, Fitchburg, WI, USA) was applied to analyze the luciferase activities at 48 h post transfection.
10
Luciferase Assay for BAMBI 3'UTR
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We constructed luciferase reporter plasmids containing the 3′-UTRs of BAMBI as well as their corresponding mutated 3′-UTRs. In detail, the pMIR-REPORT Luciferase reporter vector (Ambion, USA) was inserted with the 3′-UTRs, and mutated 3′-UTR of BAMBI. Then, 293 T cells were seeded into 96-well plates and used for luciferase report assay. Lipofectamine 3000 (Invitrogen) and reporter plasmid as well as miR-20a mimics were co-transfected into each well 24 h later. Then, the Dual-Luciferase Reporter Assay System (E1910, Promega) was applied to measure Renilla and firefly luciferase activities.
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