The 3'-UTR sequence of wild-type (wt-) CnA mRNA was amplified to the downstream site of the pGL4 luciferase vector (Promega, Madison, WI, USA). The rapid site-directed mutagenesis kit (D0206, Beyotime, Shanghai, China) was used to generate the mutated (mut-) CnA mRNA 3'-UTR. The H9c2 cells were seeded in a 24-well plate at a density of 3×104/well. After 24 hours, 1 µg of wt-CnA mRNA 3'-UTR or mut-CnA mRNA 3'-UTR luciferase plasmid, 50 nM miR-194 mimic or miR-194 NC, and 150 ng of Renilla luciferase plasmid (Beyotime, Shanghai, China) were transfected into cells via LipofectamineTM 2000. The cells were then incubated at 37 ℃ for 36 hours. The dual luciferase reporter gene detection kit (Promega, Madison, WI, USA) was used to detect luciferase activity according to the manufacturer’s protocol. All data were normalized to Renilla luciferase activity.
Renilla luciferase plasmid
Manufactured by Beyotime
Sourced in China
The Renilla luciferase plasmid is a laboratory tool used to measure gene expression. It contains the gene for Renilla luciferase, an enzyme that produces bioluminescence, which can be quantified to assess the activity of a promoter or other regulatory sequences.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter gene detection kit, by Promega (6 mentions)
Rapid site directed mutagenesis kit d0206, by Beyotime (4 mentions)
Lipofectamine 2000, by Beyotime (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Pmir report luciferase vector, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter 1000 assay system, by Promega (1 mentions)
Quickmutation kit, by Beyotime (1 mentions)
Lipofectamine 2000, by Promega (1 mentions)
8 protocols using renilla luciferase plasmid
1
Evaluating miR-194 Regulation of CnA mRNA
2
Investigating SIRT3 mRNA 3'-UTR Regulation by miR-421
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The 3’-UTR sequence of wild-type (wt-) SIRT3 mRNA was amplified to the downstream site of the pGL4 luciferase vector (Promega, Madison, WI, USA). To generate the mutated (mut-) SIRT3 mRNA 3’-UTR, the rapid site-directed mutagenesis kit (D0206, Beyotime) was used. The macrophages were seeded in 24-well plates at a density of 3 × 104/well. After 24 h, 1 µg of wt- or mut-SIRT3 luciferase plasmid, 50 nM of miR-421 mimic or miR-421 NC, and 150 ng of Renilla luciferase plasmid (Beyotime) were transfected into the macrophages via LipofectamineTM2000. The cells were incubated at 37 °C for 36 h. Following the manufacturer’s protocol, a dual luciferase reporter gene detection kit (Promega, Madison, WI, USA) was used to detect luciferase activity. All data were normalized to detect Renilla luciferase activity.
3
Validating IGF1R-miR-6838-5p Interaction
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The 3'-UTR sequence of wild-type (wt-) IGF1R mRNA was amplified to the downstream site of the pGL4 luciferase vector (Promega, Madison, WI, USA). A rapid site-directed mutagenesis kit (D0206, Beyotime Biotechnology, Shanghai, China) was used to generate the mutated (mut-) IGF1R mRNA 3'-UTR. The U2OS and HOS cells were seeded in 24-well plates at a density of 3×104/well. After 24 h, 1 µg of wt-IGF1R mRNA 3'-UTR or mut-IGF1R mRNA 3'-UTR luciferase plasmid, 50 nM miR-6838-5p mimic or miR-6838-5p NC, 150 ng of Renilla luciferase plasmid (Beyotime) were transfected into cells via LipofectamineTM 2000 (Invitrogen Corporation, Carlsbad, CA, USA) transfection reagent. The cells were then incubated at 37 °C for 36 h. According to the manufacturer’s protocol, a dual luciferase reporter gene detection kit (Promega) was used to detect luciferase activity. All data were normalized to Renilla luciferase activity. For the verification of the targeted binding of circRASSF2 and miR-6838-5p, the method was similar to the description given above.
4
Regulation of BNIP3 by miR-411-5p
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The 3′-UTR sequence of wild-type (wt-) BNIP3 mRNA was amplified to the downstream site of the pGL4 luciferase vector (Promega, Madison, WI, USA). The rapid site-directed mutagenesis kit (D0206, Beyotime, Shanghai, China) was used to generate the mutated (mut-) BNIP3 mRNA 3′-UTR. The TMD8 cells were seeded in 24-well plates at a density of 3 × 104/well. After 24 h, 1 μg of wt-BNIP3 mRNA 3′-UTR or mut-BNIP3 mRNA 3′-UTR luciferase plasmid, 50 nM miR-411-5p mimic or miR-411-5p NC, and 150 ng of Renilla luciferase plasmid (Beyotime, Shanghai, China) were transfected into cells via LipofectamineTM 2000. The cells were then incubated at 37°C for 36 h. According to the manufacturer's protocol, a dual luciferase reporter gene detection kit (Promega, Madison, WI, USA) was used to detect luciferase activity. All data were normalized to Renilla luciferase activity. For the verification of the targeted binding of LINC0461 and miR-411-5p, the method was like the above description.
5
NF-κB and AP-1 Activation in RAW264.7 Cells
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RAW264.7 cells were seeded in 24-well plates at a density of 1 × 105 cells/well and were co-transfected with 0.5 μg of NF-κB or AP-1 promoter luciferase reporter plasmid and 0.02 μg of Renilla luciferase plasmid (Beyotime, Nantong, China) using X-tremeGENE HP DNA Transfection Reagent. After 24 h, 2.0 μg/ml rCC16 was added and incubated for 2 h, after which the cells were treated with 0.1 μg/ml LPS for another 24 h. The cells were lysed, and the Firefly luciferase activity was assayed and normalized to Renilla luciferase activity using a dual luciferase reporter assay system (Promega, Madison, USA).
6
MiR-455-3p Targets Smad2 and TCONS_00039830
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The 3′-UTR sequences of wild-type (wt) Smad2 and wt-TCONS_00039830 were amplified to the downstream site of the pGL4 luciferase vector (Promega, Madison, WI, USA). The rapid site-directed mutagenesis kit (D0206; Beyotime, Shanghai, China) was used to generate the mutated (mut) SIRT3 mRNA 3′-UTR and mut-TCONS_00039830. 293T cells were seeded into 24-well plates at a density of 3 × 104 cells/well. Approximately 1 μg of wt-Smad2/mut-Smad2 or wt-TCONS_00039830/mut-TCONS_00039830 luciferase plasmid, 50 nM miR-455-3p mimic or miR-455-3p NC and 150 ng of Renilla luciferase plasmid (Beyotime) were transfected into 293T cells using the LipofectamineTM2000 reagent after 24 h, and the cells were incubated at 37 °C for 36 h. A dual-luciferase reporter gene detection kit (Promega) was used to detect luciferase activity according to the manufacturer’s instructions. All data were normalised to Renilla luciferase activity.
7
Validating miR-106a-5p Binding to LKB1 3'-UTR
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The binding sites of miR-106a-5p and LKB1 were predicted using TargetScan (http://www.targetscan.org/vert_72/ ) The 3'-UTR sequence of wild-type (WT)-LKB1 mRNA was amplified to the downstream site of the pMIR-REPORT luciferase vectors (Ambion; Thermo Fisher Scientific, Inc.). The QuickMutation™ Site-Directed Mutagenesis kit (Beijing Solarbio Science & Technology Co., Ltd.) was used to generate the mutated (MUT)-LKB1 mRNA 3'-UTR. Calu-3 and NCI-H661 cells were seeded into 24-well plates at a density of 3x104/well. After 24 h, 1 µg WT-LKB1 mRNA 3'-UTR or MUT-LKB1 mRNA 3'-UTR luciferase plasmid, 50 nM miR-106a-5p mimic or NC, and 150 ng Renilla luciferase plasmid (Beyotime Institute of Biotechnology) were transfected into cells using Lipofectamine®2000. The cells were then incubated at 37˚C for 24 h. The Dual Luciferase-Reporter 1000 Assay System (Promega Corporation) was used to evaluate luciferase activity. All data were normalized to Renilla luciferase activity.
8
Dual-Luciferase Assay for LINC00174/ENO3
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For the dual-luciferase reporter assay, 1 μg of wild-type (wt) LINC00174/ENO3-pGL4 (Promega Corporation, USA) or mutant LINC00174/ENO3-pGL4 (induced using the QuickMutation™ Kit, Beyotime, China), 50 nmol miR-2467-3p mimic/mimic NC, and 150 ng Renilla luciferase plasmid (Beyotime) were transfected into 3 × 104 cells using Lipofectamine® 2000 at 37°C for 36 h. Luciferase activity was measured using the dual-luciferase reporter gene detection kit (Promega Corporation). All data were normalized to Renilla luciferase activity.
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