According to the online bioinformatic analysis software (miRDB), the seed sequence of sja-miR-1 was predicted to be complementary with the 3'UTR of Sfrp1. To detect if Sfrp1 was a target of sja-miR-1, wild-type or mutant 3'UTRs of Sfrp1 were chemically synthesized (Genomics) and then cloned into the pmirGLO luciferase plasmid (Promega, #E1330). The 293T cells were seeded in a 24-well plate (3 × 105 cells/well). When the cells density reached up to 70%, 40 nM sja-miR-1 mimics or negative control (NC) mimics, together with 0.5 μg wild-type Sfrp1 3'UTR plasmid or mutant Sfrp1 3'UTR plasmid, were transfected into the 293T cells using lipofectamine 3000. Subsequently, the cells were cultured for 24 h and then collected. A Dual-luciferase Reporter Assay Kit (Promega, #E1901) was used to detect the effect of sja-miR-1 on the luciferase activity of the Sfrp1 3'UTR plasmid.
Pmirglo luciferase plasmid
Manufactured by Promega
Sourced in United States
The PmirGLO luciferase plasmid is a lab equipment product that provides a vector for expressing the firefly luciferase reporter gene in mammalian cells. It includes the firefly luciferase gene and a multiple cloning site for inserting target sequences.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Dual luciferase reporter detection kit, by Promega (1 mentions)
Dual luciferase reporter assay system kit, by Promega (1 mentions)
Dual luciferase kit, by Promega (1 mentions)
8 protocols using pmirglo luciferase plasmid
1
Validating Sfrp1 as sja-miR-1 Target
2
Dual-Luciferase Assay Validates circRNA-miRNA Interactions
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Interaction analysis was performed using dual-luciferase reporter assay [21 (link)]. The wild-type (WT) sequences of circ_0005526 and TCF4 3ʹUTR were inserted into pmirGLO luciferase plasmid (Promega, Madison, WI, USA), respectively. The constructed plasmids containing miR-142-5p binding sites were named as circ_0005526-WT and TCF4 3ʹUTR-WT. Then miR-142-5p binding sites in circ_0005526 and TCF4 sequences were mutated, and mutant-type (MUT) plasmids (circ_0005526-MUT and TCF4 3ʹUTR-MUT) were used as negative controls. After co-transfection with each plasmid and miR-142-5p or miR-NC for 48 h, luciferase intensity was analyzed through Dual-Luciferase Reporter Detection Kit (E1910; Promega).
3
SOS2 Luciferase Assay for miR-148a
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The SOS2 fragments harboring the predicted wild-type (WT) or mutant (MUT) binding site (Shanghai GenePharma Co., Ltd.) were cloned into the pmirGLO luciferase plasmid (Promega Corporation) to create the reporter plasmids WT-SOS2 or MUT-SOS2. NSCLC cells were co-transfected with WT-SOS2 or MUT-SOS2, together with miR-148a mimics or NC-mimics, miR-148a inhibitors or NC-inhibitors using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Relative reporter gene activity was evaluated with normalization to Renilla luciferase activity at 48 h post-transfection with a dual-luciferase reporter assay system (Promega Corporation).
4
Dual-Luciferase Assay for miR-760 Target
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The SLC9A3-AS1 fragments harboring the predicted wild-type (WT) or mutant (MUT) binding site (Shanghai GenePharma Co., Ltd.) were cloned into the pmirGLO luciferase plasmid (Promega Corporation) to create the reporter plasmids WT-SLC9A3-AS1 or MUT-SLC9A3-AS1. NSCLC cells were placed in a 24-well plate and co-transfected with WT- or MUT-SLC9A3-AS1, together with miR-760 mimic or NC-mimic using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48-h co-transfection, reporter activities were evaluated with a dual-luciferase reporter assay system (Promega Corporation, USA).
5
Luciferase Assay for miR-29b-3p
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The wild-type or mutant [UGGUGCU sequences in KDM5A 3′ untranslated region (3′UTR) binding region were mutated by random sequence] KDMA 3′UTR sequence was cloned to pmirGLO luciferase plasmid (Promega, Madison, WI, United States) via XhoI and BamHI. The luciferase plasmids were delivered into HEK293T cells with miR-29b-3p mimic or negative control (NC) mimic. The cells were harvested at 48 h after transfection and lysed with lysis buffer. The luciferase activity was detected with the Dual-Luciferase® Reporter Assay System kit (Promega, United States) on a Luminometer TD-20/20 plate reader (E5311, Promega, United States).
6
SERPINE1 3'-UTR Luciferase Assay
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The 3’-UTR (untranslated region) fragment of the wild-type SERPINE1 containing the putative miR-145 binding site and the mutant SERPINE1 sequence were cloned into pmirGLO luciferase plasmid respectively (Promega, Madison, WI, USA). HepG2 cells were transfected with different reporter vectors (pmirGLO-SERPINE1-wild-type and pmirGLO-SERPINE1-mutant) and co-transfected with the negative control (NC) or miR-145 mimic. Luciferase reporter assay was performed 48 hr after transfection using the Dual-Luciferase Kit (Promega, Madison, WI, USA).
7
Validating miR-486-5p Binding Sites
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The SLC9A3-AS1 and E2F6 fragments harbouring the predicted wild-type (WT) binding site (Shanghai GenePharma Co., Ltd.) were cloned into the pmirGLO luciferase plasmid (Promega Corporation) to create the reporter plasmids WT-SLC9A3-AS1 and WT-E2F6. Mutant (MUT) reporter plasmids, MUT-SLC9A3-AS1 and MUT-E2F6, were also constructed. NPC cells were seeded into 24-well plates and co-transfected with the reporter plasmids alongside miR-486-5p mimic or NC mimic applying Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, 48 h later, the transfected cells were lysed to detect their luciferase activity employing a dual-luciferase reporter assay system (Promega Corporation). Renilla luciferase activity was applied to normalize the firefly luciferase activity.
8
Sja-let-7 Regulates Col1α2 Expression
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To confirm Col1α2 was a target of sja-let-7, wild-type or mutant 3’UTRs of Col1α2 were chemically synthesized (GenePharma, China) and then cloned into the pmirGLO luciferase plasmid (Promega, USA). The HEK293T cells were seeded in a 24-well plate (3 × 105 cells/well). When the cells density reached up to 70%, 25 pmol sja-let-7 or NC mimics, together with 500 ng wild-type Col1α2 3’UTR plasmid or mutant Col1α2 3’UTR plasmid, were transfected into the HEK293T cells using Lipofectamine 3000. Subsequently, the cells were cultured for 48 h and then collected. A Dual-luciferase Reporter Assay Kit (Promega, USA) was used to detect the effect of sja-let-7 on the luciferase activity of the Col1α2 3’UTR plasmid.
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