Reporter assays were performed in triplicate as described in Maier et al. [26 (link)]. Schneider S2 cells (obtained from the DGRC) were transiently transfected with 1 μg of the NRE-luciferase reporter [33 (link)] and 0.2 μg of control Renilla plasmid (Promega) to normalize transfection. pMT-NICD was cotransfected with 0.5 μg of the relevant pMT-Su(H) construct and/or 0.5 μg pMT-Hairless. The total amount of transfected DNA was kept constant at 3 μg by using the pMT-A vector (Promega). Constructs were induced 6 h after transfection by adding 0.5 mM CuSO4; 18 h later, cells were harvested and luciferase activity was measured in duplicate with a luminometer (Lumat LB9507), using the dual-luciferase reporter assay system according to the manufacturer’s protocol (Promega). The effects of the addition of respective Su(H) variants relative to the controls were assessed statistically using ANOVA and Dunnett’s test (***p ≤ 0.001).
Renilla control plasmid
Manufactured by Promega
Sourced in United States
The Renilla control plasmid is a laboratory tool used to measure the expression of genes in cells. It provides a consistent reference signal for normalizing the results of gene expression experiments.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lumat lb9507, by Promega (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Luciferase reporter plasmid, by Promega (1 mentions)
Pires2 dsred express, by Takara Bio (1 mentions)
Pmir reporter construct, by Thermo Fisher Scientific (1 mentions)
Pmir report plasmid, by Thermo Fisher Scientific (1 mentions)
Expand long range dntpack, by Roche (1 mentions)
6 protocols using renilla control plasmid
1
Notch Signaling Pathway Regulation
2
Transient Transfection of 624mel Melanoma Cells
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Human 624mel melanoma cells [30] (link) were transiently transfected at approximately 90% confluency at passage three in 6-well plates. Triplicates for each construct were performed for each experiment. 2 µg of luciferase reporter plasmid (Promega) and 50 ng of control Renilla plasmid (Promega) were transfected into each well of a 6-well plate utilizing 4 µl of Lipofectamine 2000 CD reagent (Invitrogen) in Opti-MEM medium (Gibco). The cells were lysed 24 h post-transfection and Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega) with an Infinite M200 Luminometer (Tecan Munich GmbH, Kirchheim, Germany). Firefly values were divided by Renilla values to normalize for fluctuations in plated cells and transfection efficiency. Expression values of all test constructs were compared to the expression of the empty vector. Transfections were independently repeated four times per experiment. Values were transformed utilizing a transformation selector [31] (LN BASE e (X+1)) followed by a one way analysis of variance (Holm-Sidak method) (SigmaPlot v12).
3
Optimized Hypoxia Signaling Assay
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Custom-made gene synthesis was performed by Eurofins. Briefly, coral sequences were submitted to GENEius for human codon usage optimization to improve gene expression in HEK cells. A His-tag coding sequence for SpiHIFα and for SpiHIFβ coding sequence was added at the 5’ end of each gene respectively. The synthetic genes were then subcloned in pIRES2-DsRed-Express (Clontech). Both plasmids were verified by DNA Sanger sequencing.
For luciferase assays, HEK 293T cells were used because they possess high transfection efficiency. Briefly, subconfluent cells were co-transfected using Lipofectamine 2000 (Life Technologies) with 0.2 μg of HIF subunits and, 1 μg of the reporter vector (pRE-tk-LUC) containing three copies of the HRE from the erythropoietin gene [17 (link)]. The Renilla control plasmid (0.2 μg—Promega) was cotransfected with the test plasmids to control for the transfection efficiency. Luciferase activity was measured 48h post transfection.
For luciferase assays, HEK 293T cells were used because they possess high transfection efficiency. Briefly, subconfluent cells were co-transfected using Lipofectamine 2000 (Life Technologies) with 0.2 μg of HIF subunits and, 1 μg of the reporter vector (pRE-tk-LUC) containing three copies of the HRE from the erythropoietin gene [17 (link)]. The Renilla control plasmid (0.2 μg—Promega) was cotransfected with the test plasmids to control for the transfection efficiency. Luciferase activity was measured 48h post transfection.
4
Luciferase Assay for miR-221-222 Targeting
5
Evaluating Luciferase Activity in Glioma Cell Lines
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Luciferase activity was evaluated using the Dual Luciferase Reporter Assay System (Promega, USA). The human brain glioma U-87MG and U251 cell lines were transfected with TOP-flash or FOP flash plasmid (Promega, USA) along with an internal Renilla control plasmid (Promega, USA) according to the manufacturer’s protocol.
6
Cloning and Characterizing Reporter Plasmids
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