Wild-type and mutant 3′-UTR of PTEN sequences were cloned into the psi-mediated instrumental response (pMIR) luciferase reporter vector (Thermo Fisher, U.S.A.). For the luciferase assays, 100 ng pMIR luciferase reporter vector was co-transfected in cells with 100 nM miR-500a mimics or control regent, together with 20 ng pMIR-REPORT β-gal Control Plasmid (Thermo Fisher, U.S.A.) as an internal normalized control. Cells were harvested 48 h after transfection and the luciferase activities were assayed according to the manufacturer’s protocol. Transfections were performed in duplicate and repeated three times.
Pmir report β gal control plasmid
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PMIR-REPORT β-gal control plasmid is a laboratory tool used to monitor gene expression in cells. It contains the gene for the enzyme β-galactosidase, which can be detected and quantified through various assays. The plasmid is commonly used as a control or reference in experiments involving the monitoring of gene expression or the validation of reporter gene systems.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (4 mentions)
Pmir report plasmid, by Thermo Fisher Scientific (2 mentions)
Glomax 96 microplate luminometer, by Promega (2 mentions)
Mir 26a mirvana mirna mimic, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pmir reporter luciferase vector, by Thermo Fisher Scientific (1 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Pmir report luciferase plasmid, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Corning (1 mentions)
Luciferase assay system, by Promega (1 mentions)
12 well plate, by Corning (1 mentions)
Luciferase cell culture lysis reagent, by Promega (1 mentions)
Bright glo luciferase assay system, by Promega (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
High sensitivity β galactosidase assay kit, by Agilent Technologies (1 mentions)
Pmir report vector, by Thermo Fisher Scientific (1 mentions)
Pmir report mirna expression reporter vector system, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Luciferase assay system kit, by Promega (1 mentions)
9 protocols using pmir report β gal control plasmid
1
Luciferase Assay for miR-500a Regulation
2
Validating miR-185 Binding to TAZ 3'UTR
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According to results of the bioinformatics prediction, a conservative miR-185 binding sequence of the 3′untranslated region (UTR) of taz mRNA (wild-type; 5′-CAGAUGUUCUCUCC-3′) or a mutant sequence (mutant; 5′-CAGAUGAAGAGAGG-3′) was cloned as previously described (28 (link)). Luciferase reporter plasmids were generated as previously described (28 (link)) by insertion of the wild-type or mutant taz sequences into the multiple cloning site (SpeI, 5′-AAGCTT-3′ and HindIII, 5′-ACTAGT-3′) of a pMIR-REPORT™ Luciferase plasmid (Thermo Fisher Scientific, Inc.) downstream of the luciferase reporter gene. 293T cells (1×105 per well) were transfected with 0.8 µg of the luciferase constructs and 100 nM agomiR-185 or NC RNA using Lipofectamine. A total of 10 ng pMIR-REPORT™ β-gal control plasmid (AM5795; Ambion; Thermo Fisher Scientific, Inc.) was transfected as an internal control to evaluate transfection efficiency. Luminescence was measured 24 h after transfection at 37°C using a Dual-Luciferase® Reporter Assay System (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Measurements of luminescence were conducted on a Glomax 20/20 Luminometer (Promega Corporation).
3
Transfection and Reporter Assay in HD11 Macrophages
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The HD11 macrophage cells were seeded at 1.0 × 106 cells/well and cultured in 12-well plates (Corning). “Gene”/Luc were co-transfected along with miR200a/DsRed into the cells using Lipofectamine 3000 (Invitrogen) as per the manufacturer’s instructions; the pMIR-REPORT β-gal control plasmid (Ambion) was also transfected to normalize transfection efficiency. The cells were collected after 24 h and lysed by 1× luciferase cell culture lysis reagent (Promega). After centrifugation at 20 000 × g for 1 min, the supernatant was used for the measurement of the luciferase and β-galactosidase activity in 96-well plates (Corning) using Luciferase Assay Systems (Promega, Madison, WI, USA) and β-galactose solution (o-nitrophenyl-β-d -galactopyranoside or OPNG), respectively, as per the manufacturer’s instructions. β-galactosidase activities were used to normalize the reported luciferase activities. All experiments were independently replicated thrice to verify the results.
4
Luciferase Assay for miRNA Target Validation
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M17 cells were placed in 48 wells and cotransfected with 0.05 μg of the firefly luciferase report vector, 0.4 μg of hU6-hsa-miR-124-CMV-GFP or hU6-hsa-miR-Scramble-CMV-GFP, and 0.01 μg of pMIR-REPORT β-gal control plasmid (Ambion, Austin, TX, United States) for normalization of transfection using the Lipofectamine 2000 reagent according to the manufacturer’s protocol. After 24 h, the luciferase activity was measured using the Bright-Glo luciferase assay system (Promega), and the β-galactosidase activity were measured using the high-sensitivity β-galactosidase assay kit (Agilent Technologies, Palo Alto, CA, United States). The luciferase activity was normalized against the β-Gal activity for the same cells. The experiments were performed in triplicate.
5
Luciferase Assay for miR-26a Targeting
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Reporter plasmids were constructed by ligation of synthetic oligonucleotide duplexes (IDT) containing putative miR-26a target regions in the PTEN 3′UTR: 5′- CTA GTT AAC TGT TAG GGA ATT TTA CTT GAA A -3′ and 5′-AGC TTT TCA AGT AAA ATT CCC TAA CAG TTA A-3′, obtained from microRNA.org [21 ] to form a DNA duplex with overhanging SpeI and HindIII half sites in the 5′ and 3′ ends respectively, which was cloned into the appropriately digested pMIR-REPORT plasmid (Ambion). This construct was co-transfected with miR-26a mirVana miRNA mimic (Applied Biosystems) and the pMIR-REPORT β-gal Control Plasmid (Ambion) into HCT116 cells. Luciferease activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega) 48 h after transfection, in a GloMax 96 Microplate Luminometer (Promega). Luciferase activity was normalized to β-gal activity for each transfected well; each experiment was performed in triplicate.
6
PTEN 3'-UTR Regulation by miR-200c
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3′-UTR of PTEN containing predicted miR-200c binding sites was inserted into a pMIR-REPORT vector (Ambion). NP460 and HONE1 cells were co-transfected with miR-200c or anti-miR-200c, pMIR-PTEN 3′-UTR wild-type vector (Wt) or pMIR-PTEN 3′-UTR mutant vector (Mt), and pMIR-REPORT β-gal control plasmid (Ambion) using Lipofectamine 2000 (Invitrogen). Dual-Luciferase Reporter Assay System (Promega) was used to analyze luciferase activities 48 h after transfection.
7
Rb1 3'UTR miR-26a Luciferase Assay
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Reporter plasmids were constructed by ligation of synthetic oligonucleotide duplexes (IDT) containing putative miR-26a target regions in the Rb1 3′ UTR, 5′-CTA GTT AAG TAC CCA TGT AGT ACT TGA AA-3′ and 5′-AGC TTT TCA AGT ACT ACA TGG GTA CTT AA-3′, obtained from microRNA.org, 27 into the pMIR-REPORT plasmid (Ambion). This construct was co-transfected with miR-26a mirVana miRNA mimic (Applied Biosystems) and the pMIR-REPORT β-gal Control Plasmid (Ambion) into HCT116 cells. Luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega) 48 h after transfection in a GloMax 96 Microplate Luminometer (Promega). Luciferase activity was normalized to β-gal activity for each transfected well; each experiment was performed in triplicate.
8
Evaluating CDH1 3'UTR-Mediated Regulation
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JHOC-9 and OVISE cells were seeded in triplicates (or greater) in 96-well plates at densities of 1 × 104 and 9 × 103 cells, respectively. After 24 h, Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific) was used to co-transfect cells with an pMIR control luciferase vector, pMIR wild-type CDH1 3′-UTR luciferase vector (CDH1 3′-UTR wt), or pMIR mutant-type CDH1 3′-UTR luciferase vector (CDH1 3′-UTR mut; addgene, Cambridge, MA, USA) and pMIR-REPORT™ β-gal Control Plasmid (Applied Biosystems). At 24 h of post-transfection, the activities of firefly luciferase and β-galactosidase were measured using the pMIR-REPORT™ miRNA Expression Reporter Vector System (Applied Biosystems), according to the manufacturer’s instructions. All experiments were performed in triplicates, and values are presented as means ± SDs.
9
Validating miR-222 Binding in IRS1 3'UTR
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A 582-bp fragment of the wild-type 3’UTR containing the potential miR-222 binding site were inserted into a luciferase reporter vector (pMIR-REPORT Luciferase miRNA Expression Reporter Vector, AM5795, Applied Biosystems, Foster City, CA). This clone was called pMIR-IRS1-WT. To verify the binding specificity, we mutated the seed region for miR-222 from ATGTAGC to TACATCG. The synthetic mutant IRS1 3’UTR fragment was inserted into an equivalent luciferase reporter plasmid. This clone was called pMIR-IRS1-MUT. These plasmids were purchased from GenScript Co. Ltd. (Nanjing, China). For the luciferase reporter assay, the 293T cells were cultured in 24-well plates, and co-transfected with 50pmol miR-222 mimics (or miR-NC), 0.2ug luciferase plasmid (pMIR-IRS1-WT or pMIR-IRS1-MUT) and 0.2ug β-Gal plasmid (pMIR-REPORT β-gal Control Plasmid, Applied Biosystems) using Lipofectamine 2000 (Invitrogen). The β-Gal plasmid was used as a control reporter for normalization. After 48 h, the luciferase activity was measured using the Luciferase Assay System kit (E1501, Promega, Madison, WI, USA) according to manufacturer’s protocol.
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