SH-SY5Y cells were plated at a density of 1 × 105 per well in 24-well plates and transfected after 24 h with 20 nM of siRNA duplexes conjugated to 0.5 mL of Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). After 48 h from siRNA transfection, 20 ng (for PSSG-APP 3′UTR) or 30 ng (for PSSG-SERPINE 3′UTR and PSSG-KCC2 3′UTR) of firefly luciferase expression vector and 5 ng of Renilla luciferase expression vector (pRL-TK Promega, WI, USA) conjugated to 0.5 mL of Lipofectamine 2000 were transfected. After 24 h cells were lysed and luciferase assays were performed using the Dual Luciferase Reporter Assay System (Promega, WI, USA) according to the manufacturer’s protocol. The experiments were carried out in triplicate. Hippocampal neurons were plated at a density of 2 × 105 per well in 24-well plates and transfected after 72 h with 200 nM of siRNA duplexes (Invitrogen) and after 72 h from siRNA transfection with 40 ng of firefly luciferase expression vector and 4 ng of Renilla luciferase expression vector (pRL-TK Promega, WI, USA) conjugated to 2 µL of Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). After 24 h, cells were lysed and luciferase assays were performed. The experiments were carried out in triplicate.
Renilla luciferase expression vector
Manufactured by Promega
Sourced in United States
The Renilla luciferase expression vector is a laboratory tool used to express the Renilla luciferase gene in cells. It provides a means to produce the Renilla luciferase enzyme, which is commonly used as a reporter in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Probes for taqman gene expression assays, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Promega (2 mentions)
Monolight 3010 luminometer, by BD (1 mentions)
Lipofectamine plus reagent, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Pgl3 luciferase vector, by Promega (1 mentions)
Sirna duplexes, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay kit, by Promega (1 mentions)
Lipofectamin 2000, by Thermo Fisher Scientific (1 mentions)
Xbai, by Promega (1 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Superfect reagent, by Qiagen (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
10 protocols using renilla luciferase expression vector
1
Dual Luciferase Assay for Gene Regulation
2
PPAR-γ Transactivation Assay in HEK293 Cells
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Plasmid 3-x PPRE-tk-LUC containing three copies of PPAR-γ-response element and pSV-PPAR-γ2 was used (Seo et al., 2004 (link)). The pRL-SV40 construct, the Renilla luciferase expression vector, was purchased from Promega Corp. Human embryonic kidney 293 (HEK293) cells were seeded into 24-well plates and cultured for 24 h before transfection. A DNA mixture containing the PPRE-luciferase reporter plasmid (0.1 μg), pSVPPAR-γ2 (0.1 μg), and an internal control plasmid pRL-SV-40 (25 ng) was transfected using Lipofectamine transfection reagent according to the manufacturer’s recommendations. At 24 h post transfection, cells were incubated for an additional 36 h following treatment with positive control or test materials. Lucif-erase activity of cell lysates was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Relative luciferase activity was normalized to transfection efficiency using the corresponding Renilla luciferase activity.
3
Hypoxia-Inducible Luciferase Assay
4
Luciferase Assays and qRT-PCR Analysis
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Luciferase reporter assays were conducted using the Dual-Glo luciferase system co-transfected with a control Renilla luciferase expression vector (Promega) as previously reported [7 (link)]. Quantitative RT-PCR (qRT-PCR) was performed using a StepOnePlus real-time PCR system (Applied Biosystems) in conjunction with probes for TaqMan Gene Expression Assays (Applied Biosystems) according to the manufacturer's protocol.
5
Tango Assay for GPCR-Mediated Signaling
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Tango assays were performed as described previously (Barnea et al., 2008 (link)). HTL cells were transfected with the GPCR-TEV cleavage site-tTA plasmid, COMMD8-TEV protease plasmid, and pGL4.74 (hRluc/TK) Renilla luciferase expression vector (Promega) at a 10:5:1 ratio. 1 d after transfection, cells were collected in serum-free DMEM and seeded at a density of 6.4 × 104 cells per well in 96-well clear-bottom white plates. After 2–6 h of culture, cells were stimulated with CXCL12 (10 ng/ml) or isoproterenol (1 nM). Paroxetine (Merck Millipore) treatment was started 1 h before ligand stimulation. After 12 h, tTA-induced firefly luciferase activity was determined using the Dual-Glo luciferase assay system (Promega) and normalized to Renilla luciferase activity. Luminescence was measured in triplicate with the GloMax microplate reader in luminescence mode.
6
Luciferase Assay for LINC00958 3'UTR
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The 3′-untranslated region of LINC00958 was amplified using PCR and cloned into the pGL3 luciferase vector (Promega Corporation, WI, USA). A549 cells were co-transfected with firefly luciferase vector (100 ng) and Renilla luciferase expression vector (10 ng) (Promega Corporation) using Lipofectamine 2000, following the manufacturer’s instructions. At 48 h post-transfection, the luciferase reporter assay was performed using the dual-luciferase reporter assay system (Promega Corporation). Renilla luciferase (pRL-TK) served as an internal control for normalization.
7
Luciferase and qRT-PCR Assays
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Luciferase reporter assays were conducted using the Dual-Glo luciferase system co-transfected with a control Renilla luciferase expression vector (Promega) as previously reported [48 (link)]. Quantitative RT-PCR (qRT-PCR) was performed using a StepOnePlus™ real-time PCR system (Applied Biosystems) in conjunction with probes for TaqMan Gene Expression Assays (Applied Biosystems) according to the manufacturer's protocol.
8
Evaluating CLDN-3 Promoter Activity
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HEK293T cells seeded in 96-well plates were transfected with CLDN-3 promoter mimics luciferase reporter plasmid, AP-1 vector, and Renilla luciferase expression vector (Promega) using Lipofectamin 2000 (Life Technologies). Luciferase activities were measured at 24 h after transfection using a Dual-Glo Luciferase Assay kit (Promega) and firefly luciferase activities were normalized to Renilla luciferase activities.
9
Dual-Luciferase Assay for LUCAT1 and GCH1 3'-UTR
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Full-length LUCAT1 was inserted into the pGL3-basic vector (Promega Corporation) using XbaI and NdeI (cat. nos. R0145S and R0111S; New England BioLabs, Inc.) to create the LUCAT1 reporter vector. The GCH1 3'-UTR reporter vector was also constructed by insertion into the pGL3-basic vector. Using the QuickMutation™ Plus Kit (cat. no. D0208S; Beyotime Institute of Biotechnology), the binding sites 'CACUGCC' (position 869-875 in LUCAT1 or position 255-261 in the GCH1 3'-UTR) were changed to 'GCGACGG' to create mutated variants of the LUCAT1 and the GCH1 3'-UTR reporter vector. Dual-luciferase reporter assays were conducted by co-transfecting 293T cells with the Renilla Luciferase Expression Vector (Promega Corporation), the LUCAT1 or GCH1 3'-UTR reporter vector, and miR-NC or miR-34a-5p mimics using jetPRIME transfection reagent. After 48 h of transfection, the cells were lysed by the Dual-Luciferase Reporter Assay System (cat. no. E1910; Promega Corporation), and the luciferase activities were measured by GloMax® 20/20 Luminometer (Promega Corporation). Transfection efficiencies were standardised using Renilla luciferase. Table SIII shows the sequences of the primers used in this assay.
10
Tyrosinase Promoter Activity Assay in Melanoma
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To assay for tyrosinase promoter activity, B16F10 melanoma cells and HEMa-DP cells were transfected with tyrosinase reporter along with Renilla luciferase expression vector driven by the thymidine kinase promoter (Promega, Madison, WI, U.S.A.) using Superfect™ reagent (Qiagen, Valencia, CA, U.S.A.). After incubation for 24 h, cells were treated for 24 h with GAE. The cells were harvested and lysed, and the supernatants were assayed for luciferase activity using a Dual Luciferase Assay System (Promega, Madison, WI, U.S.A.).
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