Recombinant plasmids of pGL3-uMtCK-3′ UTR-wt (uMtCK-3′ UTR) or its mutant (uMtCK-3′ UTR-mut) were constructed by cloning 3′ UTR of uMtCK or its mutant into pGL3 vector (Fenghui Biotechnology). SW480 cells (50,000 cells/well) were seeded into 48-well plate, and pGL3-uMtCK-3′ UTR-wt or uMtCK-3′ UTR-mut was co-transfected with 20nM of miR-491-3p and 2 ng of pRL-TK into SW480 cells. After transfecting for 48 h, luciferase activity was examined using Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). Transfection efficiencies were normalized by renilla luciferase.
Pierce renilla firefly luciferase dual assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce™ Renilla-Firefly Luciferase Dual Assay Kit is a laboratory product that enables the simultaneous measurement of Renilla and Firefly luciferase reporter gene activity in a single sample. It provides the necessary reagents to perform this dual-reporter assay.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pgl3 vector, by Hunan Fenghui Biotechnology (1 mentions)
Pmir report vector, by Thermo Fisher Scientific (1 mentions)
Transit lt1 transfection reagent, by Mirus Bio (1 mentions)
Pgl3 luciferase promote vector, by Sangon (1 mentions)
Pmirtarget vector, by OriGene (1 mentions)
Cdk2 mrna 3 utr mutant, by GenePharma (1 mentions)
Psicheck 2 luciferase vector, by Promega (1 mentions)
Phusion site directed mutagenesis kit, by Thermo Fisher Scientific (1 mentions)
Sbe4 luc, by Addgene (1 mentions)
Synergy h1 microplate reader, by Agilent Technologies (1 mentions)
Prl sv40p, by Addgene (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
β actin clone ac 74, by Merck Group (1 mentions)
Mouse tgf beta 1 picokine elisa kit, by Boster Bio (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Pmirglo firefly luciferase reporter, by Promega (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
18 protocols using pierce renilla firefly luciferase dual assay kit
1
uMtCK 3'UTR Regulation by miR-491-3p
2
Luciferase Assay for circRNA-miRNA Interaction
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The fragments of wild type (WT) circ_0017639 and WT SIX1 3’UTR and their mutant (MUT) sequences were ligated into the pMIR-REPORT vector (Thermo Fisher). Luciferase assay was performed by co-transfecting with the luciferase vector along with miR-1296-5p or miR-NC and pRL-TK plasmids (Thermo Fisher) into DDP-resistant NSCLC cells. Analysis of luciferase activity was done with the Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (Thermo Fisher).
3
Luciferase Reporter Assay for miRNA Targets
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Sequences corresponding to the wild-type (WT) interaction sites or the mutated fragment (MUT) were inserted into the PmirGLO luciferase vector. Cells were co-transfected with the reporter plasmid and miRNA mimic or miR-NC (negative control for miRNA mimic) for 48 h. Cells were then lysed and luciferase activities were determined in the cell lysates using Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (Thermo Fisher Scientific, CA, USA).
4
CKB Regulation of Vimentin Expression
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The 1 × 104 293T cells were seeded in 96-well plate 24 h before transfection. 200ng 3xflag CKB full length or truncation plasmids together with 8ng Vimentin promoter luciferase reporter plasmid and 4ng pGL4.74 plasmids were transfected into cells using TransIT-LT1 Transfection Reagent (#Mir2300, Mirus Bio). Twenty-four to 36 h later, cells were lysed for luciferase detection using Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (#16186, ThermoFisher Scientific) according to the manufacturer's instructions.
5
Beclin-1 3'-UTR Luciferase Assay
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The 3′-UTR of beclin-1 and the mutated 3′-UTR constructs were amplified and inserted into pGL3 Luciferase Promote Vector (Sangon, Wuhan, China) with XbaI and NotI restriction sites. The pGL3 vector containing wild-type or mutated 3′-UTR of beclin-1 was cotransfected with miR-17-5p mimics (Sangon) into U87 cells with Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instruction. At 48 h after transfection, the luciferase activity of the cells was determined with the Pierce Renilla-Firefly Luciferase Dual Assay Kit (Thermo Fisher Scientific).
6
miR-148a Binding Validation on Target mRNA
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To verify miR-148a-directed binding on targeted mRNA, two 3′-UTR segments including one wild-type and the other mutant type were cloned and integrated into the pMirTarget Vector (OriGene Technologies, Inc., Rockville, MD, USA), respectively. Both the wild-type and mutant 3′-UTR segments of c-Met or ROCK1 were constructed, because c-Met and ROCK1 were predicted to be the potential target genes of miR-148a. The cells (1 × 104) were seeded in a 96-well plate for 24 h and co-transfected with two plasmids—wild-type or mutant 3′-UTR construct and pTK-Green Renilla Luc Vector (Thermo Fisher Scientific)—by using Lipofectamine 3000 (Thermo Fisher Scientific). After transfection for 48 h, a luciferase reporter assay was performed using the Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For the normalization of firefly activity, the Renilla luciferase activity was used as the internal control.
7
Validation of miR-1179 Binding Sites
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Oligonucleotides comprising the circ_0084927 mutant (the sequence containing the miR-1179 binding site was mutated to GAUACGA) or the CDK2 mRNA 3′UTR mutant (the sequence containing the miR-1179 binding site was mutated to GAUACGA) were synthesized by GenePharma (Shanghai, China). Inserted into the dual-luciferase miRNA target expression vector (pGL4) were wild-type circ_0084927, circ_0084927 mutant, CDK2 3′UTR mutant, and wild-type CDK2 3′UTR. This insertion was performed to construct luciferase reporter plasmid. HeLa and C-33A cells were also co-transfected with luciferase porter plasmid and miR-1179 mimic. After 48 h of incubation, the culture medium was removed to collect the cells. The collected cells were then lysed to obtain cell lysates. The luciferase activity was measured by Pierce Renilla-Firefly Luciferase Dual Assay Kit (16185; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the protocol.
8
Luciferase Assay Protocol for miRNA Regulation
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Luciferase assays were performed using Pierce™ Renilla-Firefly Luciferase Dual Assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Transient plasmid transfection was performed when the cells reached 70–80% confluence. The miR-498 mimic was co-transfected into C33A or SiHa cells together with MIR9-3HG-WT-1, MIR9-3HG-WT-2, MIR9-3HG-Mut-1, MIR9-3HG-Mut-2, MIR9-3HG-WT-1-WT-2, MIR9-3HG-WT-1-Mut-2, MIR9-3HG-WT-2-Mut-1 or MIR9-3HG-Mut-1-Mut-2 luciferase reporter plasmid (Promega Corporation) using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) at 37°C for 48 h. After discarding medium, the cells were washed with PBS 2–3 times. Then, 100 µl lysis buffer was added to each well and incubated in an orbital shaker in a cold room for ~20 min to fully lyse the cells. Luciferase activity was analyzed using a luminometer fluorescence detector (Turner BioSystems; Thermo Fisher Scientific, Inc.). Renilla luciferase activity was used as internal reference.
9
Validating miR-29b-3p Regulation of FOS
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FOS gene was predicted to be a target of miR-29b-3p using Tagerscan online software. For luciferase assay, the partial sequences of FOS 3′UTR containing putative miR-29b-3p binding sites were inserted into psiCHECK™-2 luciferase vector (Promega, Madison, WI, U.S.A.) to form the wile-type luciferase reporter (FOS-wt). The mutant luciferase reporter (FOS-mut) containing a point-mutated miR-29-3p binding region was generated using Phusion Site-Directed Mutagenesis Kit (Thermo Fisher). After that, constructed luciferase reporter FOS-wt or FOS-mut was transfected into CFs cells together with miR-NC or miR-29b-3p. Forty-eight hours thereafter, the cells were harvested and luciferase activity was evaluated by Pierce™ Renilla-Firefly Luciferase Dual Assay Kit (Thermo Fisher).
10
Evaluating Smad4 Signaling in Luciferase Assay
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The pCL4.26-PUMAWT and its mutant vectors were co-transfected with the pRL-Renilla luciferase control vector into Control-KD (knockdown), two Smad4-KD cell lines (#1 and #2), Control-OE (overexpression), and Smad4-OE cells. The resulting cells were cultured at 37 °C for another 24 hours and then subjected to a luciferase assay using the Pierce Renilla-Firefly Luciferase Dual Assay Kit (Thermo Fisher; #16186), following the manufacturer's guidelines.
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