For dual-luciferase reporter assays, 3′UTR of TCF21 sequence containing miR-654-5p binding sites was inserted into a pmirGLO dual-luciferase vector (Promega, USA) to generate wild-type (WT) pmirGLO-TCF21. The mutant (MUT) of TCF21 sequence in miR-654-5p binding sites was synthesized using a Site-Directed Mutagenesis Kit (F542, Thermo Fisher Scientific, USA) and inserted into a pmirGLO dual-luciferase vector to generate MUT pmirGLO-TCF21. Similarly, 3′UTR of DDR1 or MTAP containing the predicted miR-654-5p-binding sites or MUT sites was, respectively, inserted into pmirGLO dual-luciferase vector, named accordingly as pmirGLO-DDR1-3′UTR-WT, pmirGLO-DDR1-3′UTR-MUT, pmirGLO-MTAP-3′UTR-WT, and pmirGLO-MTAP-3′UTR-MUT. After that, the pmirGLO vector containing WT or MT TCF21, DDR1, or MTAP sequence was, respectively, cotransfected with miR-654-5p mimic into T/G HA-VSMC cells by Lipofectamine 2000 (Invitrogen, USA). After incubation for 48 h, the relative luciferase activity in the cells was measured using Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI).
Pmirglo dual luciferase vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PmirGLO Dual-Luciferase Vector is a reporter vector designed for monitoring gene expression in mammalian cells. The vector contains firefly luciferase and Renilla luciferase genes, enabling the measurement of both experimental and control reporter activities in a single sample.
Automatically generated - may contain errors
Lab products found in correlation
Site directed mutagenesis kit, by Thermo Fisher Scientific (4 mentions)
Pmirglo dual luciferase vector, by Promega (4 mentions)
Dual luciferase reporter assay protocol, by Promega (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
4 protocols using pmirglo dual luciferase vector
1
Dual-Luciferase Assay for miR-654-5p Targets
2
Validating miR-802 Targeting of BTF3 3'UTR
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The target-binding region of miR-802 and BTF3 was predicted using TargetScan (version 7.2; www.targetscan.org/vert_72/ ). For the dual-luciferase reporter assays, the 3′-UTR of BTF3 containing miR-802 binding sites was inserted into a pmirGLO dual-luciferase vector (Promega Corporation) to generate wild-type (WT) BTF3-3′-UTR. The mutant (mut) 3′ UTR of BTF3 was synthesized using a Site-Directed Mutagenesis kit (Thermo Fisher Scientific, Inc.) and inserted into the pmirGLO dual-luciferase vector to generate BTF3-3′-UTR-mut. The pmirGLO vector containing WT or mut BTF3 3′-UTR was co-transfected with the miR-802 mimic or NC-mimic into SiHa cells using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 48 h, the relative luciferase activity in the cells was measured using a Dual-Luciferase Reporter Assay kit (Promega Corporation) according to the manufacturer's protocol. Firefly luciferase activities were normalized to Renilla luciferase activities.
3
Validating miR-25-3p Target Sites
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The PCAT19 sequence containing miR-25-3p binding sites was inserted into the pmirGLO dual-luciferase vector (Promega, USA) to generate wild-type (WT) pmirGLO-PCAT19. The mutant (Mut) PCAT19 sequence in the miR-25-3p binding site was synthesized by using a Site-Directed Mutagenesis Kit (F542, Thermo Fisher Scientific, USA) and then inserted into the pmirGLO dual-luciferase vector to generate Mut pmirGLO-PCAT19. Similarly, the 3′ UTR of MAP2K4 containing the predicted miR-25-3p-binding sites or Mut sites was inserted into the pmirGLO dual-luciferase vector as pmirGLO-MAP2K4-3′UTR-WT and pmirGLO-MAP2K4-3′UTR-Mut, respectively. The pmirGLO vector containing the WT or Mut PCAT19 sequence and WT or Mut MAP2K4 3′ UTR was cotransfected with the miR-25-3p mimic into A549 and SK-MES-1 cells by using Lipofectamine2000 (Invitrogen, USA), respectively. After incubation for 48 h, the relative luciferase activity of the cells was measured by the Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI).
4
Dual-Luciferase Assay for miR-942 Targeting FOXA2
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For dual-luciferase reporter assay, the 3′ UTR of FOXA2 containing miR-942 binding sites were inserted into a pmirGLO dual luciferase vector (Promega, U.S.A.) to generate wild-type (WT) pmirGLO-FOXA2 3′ UTR. The mutant (MUT) 3′ UTR of FOXA2 in miR-942 binding site was synthesized using a Site-Directed Mutagenesis Kit (Thermo Fisher Scientific, U.S.A.) and inserted into a pmirGLO dual-luciferase vector to generate MUT pmirGLO-FOXA2 3′ UTR. The pmirGLO vector containing WT or MT FOXA2 3′ UTR was respectively co-transfected with miR-942 mimic into MCF-7 cells, while the pmirGLO vector containing WT or MT FOXA2 3′ UTR was co-transfected with miR-942 inhibitor into MDA-MB-468 cells by Lipofectamine2000 (Invitrogen, U.S.A.). After incubation for 48 h, the relative luciferase activities in the cells were measured by Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI).
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