For transfection of the luciferase reporter plasmid, cells were seeded in 24‐well plates, at 70%–80% confluence were washed with PBS, transfected with pGL3‐Promoter vector containing the SNP and pRL‐TK Renilla luciferase vector (Promega) as an internal control using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 48h of transfection, cell lysates were collected and operated according to the instructions of Promega's double luciferase detection kit. Firefly luciferase activity was normalized to Renilla luciferase.
Double luciferase detection kit
Manufactured by Promega
The Double Luciferase Detection Kit is a laboratory tool designed to measure the activity of two different luciferase reporter enzymes simultaneously. The kit provides the necessary reagents and protocols to quantify the luminescent signals generated by the firefly and Renilla luciferase reporter systems in a single sample.
Automatically generated - may contain errors
Lab products found in correlation
Prl tk renilla luciferase vector, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pgl3 promoter vector, by Promega (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Sinofection, by Sino Biological (1 mentions)
7 protocols using double luciferase detection kit
1
Luciferase Transfection Assay Protocol
2
Characterization of PCGEM1 3'UTR
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LINC00071 (PCGEM1) 3′UTR gene fragment was generated, and then, wild-type PCGEM1 (PCGEM1 WT) and mutant PCGEM1 (PCGEM1 Mut) were cloned into PsichecKTM-2 vector by homologous recombination method. Then, colony PCR and gene sequencing were performed to verify the success of the vector construction. Then, Lipofectamine 2000 was used to introduce the above plasmids together with miR-129-5p inhibitor and its mimics into HCT116 and SW480 cells. After 48 hours, the cells were collected and lysed. The luciferase activity of each group was detected by Promega double luciferase detection kit.
3
Regulation of 5-HT2B by miR-143 in 293T Cells
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293T cells were cultured in DMEM containing 10% fetal bovine serum (Invitrogen Corporation, USA) at 37 ℃ and 5% CO2. The cells were divided into four groups and respectively co-cultured with 5-HT2B-WT + mimic NC, 5-HT2B-WT + miR-143 mimic, 5-HT2B-MUT + mimic NC, and 5-HT2B-MUT + miR-143 mimic using a cell transfection reagent (Invitrogen Corporation, USA) according to the manufacturer’s protocol. The miRNAs were synthesized by Sangon Biotech (Shanghai) according to the specific sequences (Table 3 ). After 48 h of co-culturing, the cell culture medium was removed and the reporter gene cell lysate was added (FENGHUISHENGWU, Hunan). After the cells were fully lysed, the luciferase activity was detected using a double luciferase detection kit (Promega Corporation, Madison, WI) using a multifunctional enzyme labeling instrument with a chemiluminescence detection function. The experiments were repeated three times.
4
Regulation of miR-33a-5p promoter activity
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Cancer cells (6x103/well) were seeded in six-well plates and luciferase vectors containing the miR-33a-5p promoter (miR-33a-5p-luc) were co-transfected with TRIB2 and c-Fos expression vectors using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h of transfection, the cells were collected and lysed on ice. The luciferase activity was detected according to the manufacturer's instructions using a double luciferase detection kit (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity. Targetscan7.1 (http://www.targetscan.org/vert_71/ ) was used to predict the downstream target genes of miR-33a-5p.
5
Evaluating β-catenin Signaling and TMEM119 Promoter Activity
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To detect β-catenin activity, cells were seeded into 24-well plates at 8 × 104 cells/well. A responsive vector of β-catenin signaling TopFlash or A non-responsive vector FOPFlash and the Sea Kidney Luciferase expressed by pRL-TK PRL-TK plasmid were co-transfected into 293 T cells using Sinofection® (SinoBiological). For examining TMEM119 promoter activity, the promoter sequences of TMEM119 containing β-catenin binding sites or not were inserted into pGL3-promoter vector, denoted as pGL3-TMEM119-wt and pGL3-TMEM119-mut. Then pGL3-TMEM119-wt or pGL3-TMEM119-mut was co-transfected with PRL-TK plus β-catenin overexpression or knockdown. After 72 h of transfection, the double luciferase detection kit (Cat # E1910, Promega) was used to measure the luciferase activity. The results were normalized to PRL-TK plasmid activity.
6
Analyzing TSPAN8 Promoter Activity
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At 24 hours before transfection, the cells in the logarithmic growth were inoculated into a 48-well plate at 2×104/well concentration. TopFlash (a responsive vector of β-catenin signaling) or FOPFlash (a non-responsive vector of β-catenin signaling) and the PRL-TK plasmid (Sea Kidney Luciferase Expressed by pRL-TK) were co-transfected into cells using Lipofectamine 2000 transfection reagent. For validating the promoter activity of TSPAN8 promoter, the promoter sequences of TSPAN8 with or without β-catenin binding sites were inserted into pGL3-promoter vector, named as pGL3-TSPAN8 and pGL3-TSPAN8-mut. The transfection procedure is same with the aforementioned process. After 72 hours of transfection, the luciferase activity was detected by Promega double luciferase detection kit. The results were standardized by comparing with the activity of PRL-TK plasmid.
7
Anxa2 Promoter Regulation by p-STAT3
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The promoter sequence of Anxa2 and the potential binding site of p-STAT3 was obtained from the JASPAR database (http://jaspar.genereg.net/ ). The Anxa2 promoter sequence integrating this site was inserted in the pGL4-Luc-Report vector (Promega, Shanghai, China) to construct the promoter luciferase reporter. The above vectors were transfected into hepatocytes. Then, the cells were treated with DMSO or STAT3-specific inhibitor APTSTAT3-9R. After 48 h, the luciferase activity was determined by a double luciferase detection kit (Promega).
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