Wild type segments of the 3′UTR of MDC1 containing putative miR-22 binding sites and deletion mutants of predicted miR-22 binding sites were cloned into pMIR-REPORT firefly luciferase vector (Applied Biosystems) as described previously [17 (link)]. For the luciferase activity assay, pMIR-REPORT luciferase vectors containing wild type or mutant 3′UTRs of DNA-PKcs and pRL-TK vector containing Renilla luciferase as a transfection control were co-transfected into MCF-7 cells using Lipofectamine 2000 (Invitrogen), and subsequently, the same cells were treated with 100 nM TPA. After 3 days, the luciferase assay was performed using the dual luciferase reporter assay system (Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. Luciferase activity was quantified using a luminometer (Glomax, Promega). The luciferase activity data were normalized to the Renilla value, and the results were represented as the average and standard deviation (SD) from triplicate of experiments.
Pmir report firefly luciferase vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PMIR-REPORT firefly luciferase vector is a plasmid DNA construct that contains the firefly luciferase gene. The firefly luciferase gene serves as a reporter gene, which can be used to quantify gene expression in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Glomax, by Promega (3 mentions)
Geneart site directed mutagenesis kit, by Thermo Fisher Scientific (2 mentions)
Turbofect, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Luciferase reporter assay kit, by Abcam (1 mentions)
8 protocols using pmir report firefly luciferase vector
1
Luciferase Assay for miR-22 Binding
2
Regulation of FANCM by miR-146a
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Segments of the 3′UTR of FANCM containing putative miR146a binding sites were cloned into pMIR-REPORT firefly luciferase vector (Applied Biosystems, CA). Deletion mutants of predicted miR146a binding sites were made using the GENEART Site-Directed Mutagenesis kit (Invitrogen). For the luciferase activity assay, pMIR-REPORT luciferase vectors containing wild type or mutant 3′UTRs of FANCM and pRL-TK vector containing Renilla luciferase as a transfection control were co-transfected into HeLa cells using Lipofectamine 2000 (Invitrogen), and subsequently, the same cells were transfected with miR146a, anti-miR146a or RelA/p65 expression construct. After 24 h of transfection, the luciferase assay was performed using the dual luciferase reporter assay system (Promega, WI) according to the manufacturer's instructions. Luciferase activity was quantified using a luminometer (Glomax, Promega).
3
Vimentin 3'UTR Luciferase Assay
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The partial 3′-UTR of human vimentin was amplified from cDNA via PCR and cloned into the SpeI/MluI sites of pMIR-REPORT firefly luciferase vector (Applied Biosystems). The putative miR-124-3p and miR-138-5p binding sites within vimentin 3′-UTR were amplified via PCR and validated by sequencing. The miR-124-3p- and miR-138-5p-overexpressing and control cells were seeded and co-transfected with reporter plasmids and the pSVβ vector (Clontech Laboratories, Inc., Mountain View, CA, USA) using TurboFectTM (Fermentas, Glen Burnie, MD, USA). After transfection for 24 h, luciferase activity was measured and compared with that of the control group. Luciferase activity was normalized to that of β-galactosidase.
4
Identifying miR-145 Binding Sites in DNA-PKcs 3'UTR
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Segments of the 3′UTR of DNA-PKcs containing putative miR-145 binding sites were cloned into pMIR-REPORT firefly luciferase vector (Applied Biosystems, Waltham, MA, USA). Deletion mutants of predicted miR-145 binding sites were made using the GENEART Site-Directed Mutagenesis kit (Invitrogen). For the luciferase activity assay, pMIR-REPORT luciferase vectors containing wild type or mutant 3′UTRs of DNA-PKcs and pRL-TK vector containing Renilla luciferase as a transfection control were co-transfected into HeLa cells using Lipofectamine 2000 (Invitrogen), and subsequently, the same cells were transfected with miR-145 and/or anti-miR-145. After 24 h of transfection, the luciferase assay was performed using the dual luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Luciferase activity was quantified using a luminometer (Glomax, Promega).
5
Luciferase Reporter Constructs for EGR1, CITED2, and BMP7
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Luciferase reporter constructs containing nucleotides 1 to 283 bp of the EGR1 5′UTR and 1916 to 3137 bp of the EGR1 3′UTR, 1 to 244 bp of the CITED2 5′UTR and 1058 to 2382 bp of the CITED2 3′UTR, 1 to 519 bp of the BMP7 5′UTR, 1816 to 2950 BMP7 3′UTR, and 2901 to 4021 BMP7 3′UTR, wild-type 5′UTR of the EGR1 5′UTR and mutants of 227 to 267 bp in the EGR1 5′ UTR binding site, wild-type 5′UTR of the CITED2 3′UTR and mutants of 2045 to 2087 bp in the CITED2 3′UTR binding site, and wild-type 5′UTR of the BMP7 5′UTR and mutants of 175 to 225 bp in the BMP7 5′ UTR binding site were cloned into PGL4-RLuc luciferase vector (Promega, Madison, WI, USA) or pMIR-REPORT firefly luciferase vector (Ambion, Austin, TX, USA). In luciferase reporter experiments, HSFBs were transfected with 400 ng of luciferase plasmid (containing the wild-type UTR binding site) or mutation luciferase plasmid (containing the corresponding UTR mutant) in a final volume of 0.2 ml using Lipofectamine 2000 (Invitrogen). At the same time, control or experiment vector/regents were cotransfected into HSFBs and treated with TA and stretch. The mRNA expression of luciferase was determined by qRT-PCR. The primers used for fragment cloning and PCR were listed in table S1.
6
Luciferase Assay for SPATS2 and GLE1 3′ UTRs
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Luciferase reporter constructs containing regions of the SPATS2 (NM_001293286.1) and GLE1 (NM_001003722) 3′ UTRs were cloned into pMIR-REPORT firefly luciferase vector (Ambion). pRL-Tk Renilla luciferase reporter was used as transfection control and luciferase assay normalization. The assays were performed 48 h after transfection of the indicated constructs into 2.4 × 104 HEK293 cells per well (four wells each samples) seeded in 96-well plates. The cells were transfected with 50 ng of firefly luciferase vectors and 1 ng of the pRL-Tk Renilla reporter. The firefly reporters were competed by co-transfecting 50 ng of pcDNA 3.1 (+) expressing the SPATS2 cDNA. The reporter activities were measured using the Dual Glo Luciferase assay System (Promega) and GloMax Multi Detection System (Promega). The following primers were used for the fragment subcloning PmeI–HinIII into pMIR-REPORT: SPATS2 3′ UTR forward PmeI primer 5′-GTTTAAACGCCTCTATCCCAGAATGTGC-3′ and reverse HindIII primer 5′-AAGCTTTGCTACTTTCTCATAGACTTCCCTA-3′; GLE1 3′ UTR forward PmeI primer 5′-GTTTAAACCCTCCCTTGTCTCTAGTGTCTT-3′ and reverse Hind III primer 5′-AAGCTTCAACCATAACCCAGACTTGCAT-3′.
7
Luciferase Assay of MDM2 3'UTR
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The wild-type (WT) and mutant 3′-untranslated region (3′-UTR) of MDM2, containing the predicted binding site of miR-188-3p, were constructed into the pMIR-REPORT firefly luciferase vector (Ambion; Thermo Fisher Scientific, Inc.). For the luciferase experiments, cells were seeded into the 96-well plate at the density of 1,000 cells/well. Cells were then co-transfected with 100 nM miR-188-3p or NC miRNA in the presence of 50 µg luciferase vector containing either WT or mutant forms of the 3′-UTR of MDM2 using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.). Following transfection for 36 h, the luciferase activity was determined and normalized with the activity of Renilla luciferase using a Luciferase Reporter assay kit (BioVision, Inc., Milpitas, CA, USA). The experiment was performed in triplicate.
8
Constructing Translational Reporters for PDCD4 and CX43
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pMIR-REPORT firefly luciferase vector was purchased from Ambion (Austin, TX, USA). pRL-TK Renilla luc reporter was obtained from Promega (Madison, WI, USA).
Complementary DNA clones containing fragments of the 3′ UTR of PDCD4 (clone ID: NM_014456) and CX43/GJA1 were purchased from Open Biosystems and OriGene (Rockville, MD, USA), respectively.
To construct a WT PDCD4 translational reporter, a 1.7-kb fragment representing the 3′ UTR was excised using MluI and inserted into MluI-digested pMIR-REPORT. To construct a WT CX43/GJA1 translational reporter, a 1.7-kb fragment representing the CX43 3′ UTR was generated by sequential treatment with EcoRI, Klenow fragment and MluI. This fragment was inserted into pMIR-REPORT vector that was prepared by sequential treatment with SacI, Klenow fragment and MluI.
PDCD4 and CX43 reporters with mutation in the miR-seed complementary regions were generated by PCR mutagenesis. Oligonucleotides are listed inSupplementary Table S1 . WT reporters were mutated so as to conserve the predicted secondary structure of the 3′ UTR.89 (link) Cloned PCR products were confirmed by sequence analysis.
Complementary DNA clones containing fragments of the 3′ UTR of PDCD4 (clone ID: NM_014456) and CX43/GJA1 were purchased from Open Biosystems and OriGene (Rockville, MD, USA), respectively.
To construct a WT PDCD4 translational reporter, a 1.7-kb fragment representing the 3′ UTR was excised using MluI and inserted into MluI-digested pMIR-REPORT. To construct a WT CX43/GJA1 translational reporter, a 1.7-kb fragment representing the CX43 3′ UTR was generated by sequential treatment with EcoRI, Klenow fragment and MluI. This fragment was inserted into pMIR-REPORT vector that was prepared by sequential treatment with SacI, Klenow fragment and MluI.
PDCD4 and CX43 reporters with mutation in the miR-seed complementary regions were generated by PCR mutagenesis. Oligonucleotides are listed in
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