All stable transfection were completed using Effectene transfection reagent (Invitrogen). Human embryonic kidney 293T (HEK293T) cells were seeded in 12-well plates and were transiently added LV-miR-124 and reporter gene plasmids. The cells were harvested for 48 h and luciferase activity was measured using a dual reporter assay kit (Promega, WI, USA) according to the manufacturer's instructions. The Renilla luciferase activity was used as normalization.
Dual reporter assay kit
Manufactured by Promega
Sourced in United States
The Dual Reporter Assay Kit is a laboratory tool used to measure the activity of two different reporter genes simultaneously within the same cell or sample. The kit provides reagents and protocols for the quantitative analysis of firefly and Renilla luciferase reporter activities.
Automatically generated - may contain errors
Lab products found in correlation
Cell culture lysis buffer, by Promega (5 mentions)
Glomax luminometer, by Promega (3 mentions)
Hd xtreme transfection reagent, by Roche (2 mentions)
Renilla tk luciferase vector, by Promega (2 mentions)
Effectene transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lb960 luminometer, by Berthold Technologies (1 mentions)
Prl tk renilla luciferase vector, by Promega (1 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Glomax discover luminometer, by Promega (1 mentions)
Infinite 200 pro luminometer, by Tecan (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Prl sv40, by Promega (1 mentions)
15 protocols using dual reporter assay kit
1
Transient Transfection and Luciferase Assay
2
Measuring IRES activity in MDA-MB 231 cells
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cDNA coding for the A/B (amino acids 2–173) domain of the human ESR1 gene encoding ERα was amplified by polymerase chain reaction (PCR) and cloned into the SpeI and NcoI sites of the pTRIP CRF1AL2 bi-cistronic vector that encodes both the Renilla luciferase (LucR) and Firefly luciferase (LucF2CP) genes separated by this putative IRES-ERα sequence [24 (link)]. The final construct was verified by sequencing. In such a transgene, LucR expression is cap-dependent whereas LucF expression is IRES-dependent; thus, the level of IRES activity can be deduced from the LucF/LucR ratio. The production of lentiviral particles was performed in HEK293 cells. Transduced MDA-MB 231 cells (MDA-A/B) were subjected to ER stress as indicated. To test whether the stress-induced increase in LucF activity was not due to the generation of mono-cistronic LucF transcripts via an internal promoter or cryptic splicing, MDA-Lenti-AB (1/10) cells were exposed to two siRNAs-lucR and treated with 5 mM DTT or 100 nM thapsigargin. As control, cells were treated with scrambled siRNA. After a PBS wash, cells were frozen at –80 °C. Luciferase measurements were performed with a LB960 luminometer (Berthold) using the dual reporter assay kit (E1960; Promega) according to the manufacturer’s recommendations.
3
Dual Luciferase Assay for Promoter Activity
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Cells were transfected by electroporation with the indicated promoter reporter plasmids together with pTk-Renilla for normalisation. Cells were stimulated with the indicated agents 24 h post-transfection followed by lysis in Promega cell culture lysis buffer. Luciferase and Renilla activity was quantified using the dual reporter assay kit (Promega) according to the manufacturer's instructions using Glomax luminometer (Promega).
4
BMP4-responsive Hepcidin Promoter Assay
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HepG2 cells were transiently transfected with pGL2-Luc-HAMP vector harbouring BMP-responsive hepcidin promoter upstream to Firefly luciferase reporter gene (kindly provided by M. Poli, Molecular Biology Laboratory, DMMT, University of Brescia), in combination with pRL-TK Renilla Luciferase vector (Promega) as a control for uniform lysate analyses. 12 hours after transfection, serum-starved cells were stimulated with 50 ng/mL of BMP4 (R&D) for 16 hours in the absence or in the presence of increasing concentrations of gremlinWT or gremlinC141A. Cells were then lysed and luciferase activity was determined with the Dual Reporter Assay kit (Promega). Relative luciferase activity was calculated as the ratio of Firefly (reporter) to Renilla (control) luciferase activity and is expressed as percent of the activity of BMP4-stimulated cells.
5
TM4SF1 Transcriptional Regulation Assay
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Cells were seeded at a density of 2 × 104 cells/well in a 24‐well plate. After 18 hours, cells reached 80% confluence. For each well, cells were transfected with 0.5 µg of TM4SF1 expression vector, 0.05 µg of Topflash and 0.01 µg of TK Renilla. The Fopflash was used as the control. After 24 hours, the reporter activity was measured using a dual reporter assay kit (Promega).
6
Quantifying CREB Activity in VSMCs
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CREB activity was determined by quantifying the luciferase reporter activity in cells transfected with CREB-LUC (α-168), which contains 168 bp of the endogenous alpha polypeptide glycoprotein hormones gene (CGA) promoter containing two identical repeats of the 8-bp core CRE52 (link),53 (link). Plasmid transfection was performed by electroporation using an Amaxa Nucleofector-1.5. 1 × 106 VSMCs were transfected with 5 μg of DNA or 100 pmoles of siRNA using the standard Nucleofector program A033. Cells were stimulated with the indicated agents 24 hours post transfection followed by lysis in Promega cell culture lysis buffer or for secreted nano-luciferase reporters cell culture media collected and assays using NanoGlow assay system (Promega). Luciferase and Renilla activity were quantified using the dual reporter assay kit (Promega) according to the manufactures instructions using Glomax Discover luminometer (Promega).
7
Liposome-Mediated Transfection of miR-34a and Axl 3'UTR
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A liposome‐mediated transfection method was used. The experimental groups were pmirGLO+ miR‐NC + pRL‐tk, pmirGLO+ miR‐34a + pRL‐tk, pmirGLO‐Axl 3′ UTR+ miR‐NC + pRL‐tk, and pmirGLO‐Axl 3′ UTR+ miR‐34a + pRL‐tk. Three replicate experiments were performed for each group. PmirGLO was the negative control for pmirGLO‐Axl 3′ UTR, miR‐NC was the negative control for miR‐34a, which was transfected at the same time, and pRL‐tk (sea kidney luciferase) was used as an internal control. Specific transfection methods were in accordance with the Lipofectamine 2000 reagent instructions. Luciferase activity was detected by the dual‐reporter assay kit from Promega, and the instrument for measuring the chemiluminescence resulting from the assay was a 30IOC chemiluminescence analyzer.
8
Dual-Luciferase Assay for Transcriptional Activity
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Cells were transfected by electroporation with the indicated SRE and CCN1 promoter reporter plasmids together with pTk-Renilla for normalisation. Cells were stimulated with the indicated agents 24 h post-transfection followed by lysis in Promega cell culture lysis buffer. Luciferase and Renilla activity were quantified using the dual reporter assay kit (Promega) according to the manufacturer's instructions using Glomax luminometer (Promega).
9
Dual-Luciferase Assay for Transcriptional Activation
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Cells at 70% confluence were plated in a 24-well plate. For each well, the cells were transfected with 0.01 µg of TOPFlash, 0.01 µg of TK Renilla, and 0.25 µg of DSTYK expression vector or empty vector. Forty-eight hours later, the cells were lysed with RIPA buffer, and reporter activity was examined using a dual reporter assay kit (Promega).
10
Transfection and Luciferase Assay for GR Promoter
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Transfection of plasmid DNA was performed with the aid of HD Xtreme transfection reagent (Roche) as previously described (Mata-Greenwood et al. 2013 ). Briefly, confluent cells were transfected with the GR promoter/pGL3-luciferase constructs, using a 1:1 complex of DNA:Xtreme reagent according to the manufacturer’s protocol. TK-Renilla luciferase vector (Promega Corp.) was used as the internal control. The transfection was carried out at 37 °C for 6 h. The cells were allowed to recover in a complete culture medium for 18–20 h, and then treated with starvation media with or without DEX (0.2 and 1 µM) for another 24 h, before adding passive lysis. Firefly and Renilla luciferase activities were measured using a dual-reporter assay kit (Promega) according to the manufacturer’s protocol. Relative luciferase values were calculated as a ratio of firefly/renilla luciferase activities. Each treatment was tested in quadruplicates and averages per sample were used to calculate group averages and standard errors.
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