The wild-type (wt) 3′-UTR TMPO-AS1 was amplified and cloned into pmirGLO reporter (Promega) to construct TMPO-AS1-wt plasmid. TMPO-AS1 mutant 3′-UTR plasmid (TMPO-AS1-mut) was constructed by using a TaKaRa MutanBEST kit (TaKaRa). Then, the report vector and miR-320a were cotransfected into HCCLM3 and SNU-387 cell by utilizing Lipofectamine 2000 (Thermo Fisher Scientific). The wild-type (wt) 3′-UTR SERBP1 was amplified and cloned into pmirGLO reporter (Promega) to generate SERBP1-wt plasmid. SERBP1 mutant 3′-UTR plasmid (SERBP1-mut) was constructed by utilizing a TaKaRa MutanBEST kit (TaKaRa). Then, the report vector and miR-320a were cotransfected into HCCLM3 and SNU-387 cell using Lipofectamine 2000 (Thermo Fisher Scientific). After 48 h, the luciferase activities in cells were detected by Luciferase reporter gene system (Promega).
Pmirglo reporter
Manufactured by Promega
Sourced in United States
The PmirGLO reporter is a versatile and reliable tool for gene expression analysis. It features a dual-luciferase reporter system that allows for the simultaneous measurement of two different gene expression activities within the same sample. The PmirGLO reporter is designed to provide accurate and reproducible results, enabling researchers to effectively study gene regulation and perform various experimental applications.
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 (4 mentions)
Modulus single tube multimode reader, by Promega (2 mentions)
Dual luciferase reporter assay system kit, by Promega (2 mentions)
Mutanbest kit, by Takara Bio (2 mentions)
Dual luciferase reporter system, by Promega (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pmirglo reporter vector, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Takara mutanbest kit, by Takara Bio (1 mentions)
11 protocols using pmirglo reporter
1
Luciferase Assay for miR-320a Targets
2
Luciferase Assay for PACS1 3'-UTR
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The wild-type (Wt) or mutant (Mut) sequence of PACS1 3′-untranslated region (3′-UTR) was inserted into pmiRGLO reporters (Promega, Madison, WI, USA). PACS1-Wt or PACS1-Mut was co-transfected, respectively, with miR-485-5p mimics or NC mimics into pericytes. Lipofectamine 2000 was applied to perform the transfection. The luciferase assay was carried out employing a dual-luciferase reporter assay system kit (Promega) after 48 h of transfection. The luciferase activity was evaluated by the Modulus single-tube multimode reader (Promega).
3
Luciferase Assay for CDKN1B 3'UTR
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The wild type or mutant sequence of CDKN1B 3ʹUTR was subcloned into the pmirGLO reporters (Promega, USA), and named CDKN1B-Wt or CDKN1B-Mut, respectively. Next, CDKN1B-Mut or CDKN1B-Wt was cotransfected with NC mimics or miR-221-3p mimics in 16HBE cells. Lipofectamine 2000 was employed to perform the transfection. After 48 h of transfection, a dual-luciferase reporter assay system kit (Promega) was used to conduct the luciferase assay. Modulus single-tube multimode reader (Promega) was used to detect luciferase activity.
4
Regulation of MAPK1 by miR-543
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Wild type or mutant sequences of MAPK1 3′ UTR were cloned into pmirGLO reporters (Promega, United States). MAPK1 3′ UTR-Wt or MAPK1 3′ UTR-Mut vectors were cotransfected with miR-543 mimics or NC mimics, respectively, into ESCs. Lipofectamine 2000 (Invitrogen) was used for the transfections. Forty-eight hours later, luciferase reporter assays were used to examine the relative luciferase activities. Each biological sample was run in triplicate and experiments were independently repeated three times.
5
Validation of miR-28-5p Binding Site
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The sequence of TUC38 with wild-type or mutant miR-28-5p binding site was synthesized and inserted into pmirGLO reporter (Promega, WI, USA). Then, cells were transfected with miR-28-5p mimics and wild-type or mutant pmirGLO reporter by using Lipofectamine 2000 (Invitrogen). The transfection efficiency was detected by qRT-PCR assay. After 48 h, the luciferase activity in each well was measured using the Dual-Luciferase Reporter System based on supplier's instructions (Promega).
6
Regulation of CCR7 by let-7e-5p
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The wild type (WT) and mutant type (MT) of CCR7 3'UTR sequences were generated by PCR amplification from human genomic DNA using specific primers (Table 2 ) and cloned into pmirGLO- reporter (Promega), respectively. PCI-37B cells were transfected with either plasmid for the WT or MT of CCR7 3' UTR luciferase reporter, together with control scramble or hsa-let-7e-5p mimic as well as the plasmid for Renilla luciferase. Two days later, the luciferase activities were measured using Dual Luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalized to the renilla luciferase activity.
7
miR-142a-3p Regulation of Rab3a mRNA
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Potential target genes of miR-142a-3p were predicted using online software, including Targetscan Mouse (http://www.targetscan.org/ ), MicroCosm Targets (http://www.ebi.ac.uk/enright-srv/microcosm/ ), and Miranda (http://mirdb.org/ ). Among the candidate target genes, Rab3a contains a conserved mRNA 3′UTR sequence element that is predicted to be complementary to the seed region of mouse miR-142a-3p. The Rab3a mRNA 3′UTR sequence was amplified from mouse brain by RT-PCR, and the mutation in the miR-142a-3p binding site was amplified using overlap PCR. The PCR-amplified wild-type Rab3a mRNA 3′UTR (Rab3a-WT) and the mutated Rab3a mRNA 3′UTR (Rab3a-MUT) were then cloned into the firefly luciferase pmirGLO reporter (Promega, Madison, USA) using the NheI and XhoI sites. The Dual-Luciferase Reporter Assay System (Promega, Madison, USA), with firefly luciferase (luc2) as the primary reporter was used to monitor the interaction of miR-142a-3p and Rab3a mRNA 3′UTR. Renilla luciferase (hRluc-neo) was used as a control reporter for normalization and selection. HEK293T cells were co-transfected with Rab3a-WT/Rab3a-MUT, miRNA mimic/inhibitor/NC, and the Renilla luciferase construct, and the luciferase activity in these cell lysates was measured at 24 h post-transfection.
8
Regulation of Ulk1 by miR-142-5p
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The rat Ulk1 3′UTR was amplified by RT-PCR from mouse brain cDNA (P15). Mutation of the miR-142-5p binding site was achieved using the Multisite-Quickchange kit (Stratagene, USA) according to the manufacturer's protocol. To further confirm the regulation of Ulk1 by miR-142-5p, the luciferase pmirGLO reporter (Promega, Madison, USA) was constructed and then confirmed by sequencing. Luciferase activity was detected 48 h after the co-transfection of the luciferase construct (with either wild-type or mutant-type miR-142-5p binding sites), the miR mimics/inhibitors or control (RiboBio, Guangzhou, China), and a Renilla luciferase vector in HEK293T cells. The Dual-Luciferase Reporter Assay System (Promega, Madison, USA) was used to quantify the effects of a miR-142-5p interaction with the Ulk1 3′UTR.
9
Luciferase Assay for miRNA Binding
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The wild-type (WT) RCAN1 3′-UTR sequence was amplified and cloned into the pmirGLO reporter (Promega) to generate the RCAN1-WT plasmid vector. The TaKaRa MutanBEST kit (TaKaRa) was handled to construct mutant (MUT) RCAN1 3′-UTR plasmid vector (RCAN1-MUT). Then, the RCAN1-WT/MUT reporter vector and miR-mimic/miR-NC were cotransfected into Hep3b cells with Lipofectamine 2000 (Thermo Fisher Scientific). After 48 h of transfection, luciferase activities of cells were assessed by the luciferase reporter gene system (Promega).
10
Luciferase Reporters for miRNA-mRNA Interactions
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Luciferase reporters were designed and reporter assay was performed as previously described.20 (link) The association between LINC00665 and miR-1224-5p or between miR-1224-5p and SND1 was tested by using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). In brief, 5000 DU-145 cells per well were seeded into 24-well plates and transfected with pmirGLO reporter (Promega) containing wild-type (wt) or mutant (mut) LINC00665 or SND1-3ʹ-UTR region and miR-1224-5p mimics using Lipofectamine 3000 reagent (Invitrogen). After 48h, luciferase activity was determined according to the manufacturer’s protocols.
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