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Prl tk renilla luciferase reporter vector

Manufactured by Promega
Sourced in United States, China

The PRL-TK Renilla luciferase reporter vector is a plasmid that contains the Renilla luciferase gene under the control of a thymidine kinase (TK) promoter. Renilla luciferase is a bioluminescent reporter enzyme that can be used to measure gene expression or other cellular events in a variety of experimental systems.

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54 protocols using prl tk renilla luciferase reporter vector

1

HEK-293T Cell Transfection Assay

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Human Embryonic Kidney (HEK) -293T cells (provided by the Department of Biochemistry, Guangdong Medical, China) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 100 mg streptomycin and 100 U/ml penicillin at 5% CO2 and 37°C. Cells were seeded in 24-well plates at approximately 2×105 cells per /well before transfection. Cells were transiently transfected using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocol. For each cell, 0.9 g of the construct was co-transfected along with 0.1 g of pRL-TK renilla luciferase reporter vector (Promega) to control transfection efficiency and variation in plating. All the experiments were performed in three replicates for each construct.
Approximately 48 h after transfection, cells were harvested and the activity of both firefly and Renilla luciferases were measured using a Dual-Luciferase Reporter Assay System (Promega) on a Modulus microplate multimode reader (Turner Biosystems, CA, USA). The normalized luciferase data (firefly/renilla) were used to perform the average statistics of three replicates.
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2

Measuring Wnt/β-Catenin Signalling Activity

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Wnt/β-catenin signalling activity was measured using a β-catenin firefly luciferase reporter plasmid containing TCF/LEF binding sites (TOP flash) (Addgene, Cambridge, MA, USA, Catalog #12456). The β-catenin reporter plasmid containing mutated TCF/LEF binding sites (FOP flash) (Addgene, Catalog #12457) served as negative control. As described previously,32 (link) cells were transfected with β-catenin reporter plasmid and pRL-TK renilla luciferase reporter vector (Promega, Fitchburg, WI, USA, catalog #E2241), as an internal control for transfection efficiency, using the Lipofectamine LTX reagent (Life Technologies, catalog #15338030). Simultaneously, siRNA and pENTR4 plasmid vector were respectively transfected into CRC cells and CCD841 CoN cells. After transfection, cells were maintained in serum-free conditions for 2 to 3 days until they were examined using a Dual-luciferase Reporter Assay system kit (Promega, catalog #E1910) and a microplate reader (Corona Electric, Lethbridge, Canada, catalog #SH-9000), following the manufacturers’ instructions. Reporter activities were reflected by the luminescence intensity. Data were normalised by the value of pRL-TK renilla luciferase activity.
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3

MGMT Luciferase Reporter Assay

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Constructs of the MGMT firefly luciferase reporter or their mutants were transfected or cotransfected with the siRNAs, into cells for 48 h. The pRL‐TK Renilla luciferase reporter vector was severed as an internal control (Promega) to normalize firefly luciferase activity. Luciferase activities were measured using the dual‐luciferase reporter assay system (Promega).
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4

Luciferase Assay for miRNA Binding

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The pGL3 vector (Promega, Madison, SUA) was used to construct the reporter vectors, and the fragment of wild‐type 3′UTR of FOXO3 or PERP containing the putative miR‐629 binding site was amplified from human genomic DNA and cloned into the downstream of the pGL3 vector, For the mutant, point mutations were introduced into the seed region of miR‐629 bindings sties. For the luciferase reporter assay, cells were co‐transfected with wild‐type or mutant reporter vectors, respective miRNAs (mimics NC or miR‐629 mimics) and pRL‐TK renilla luciferase reporter vector (Promega) by Lipofectamine 200 reagent (Invitrogen) according to the manufacturer's protocol. Relative luciferase activities were determined by Dual‐Luciferase Reporter Assay system (Promega) at 48 hours after co‐transfections.
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5

Validating miRNA-Target Gene Interactions

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Using the TargetScan 8.0 database (http://www.targetscan.org/, accessed on 1 January 2020), the binding sites of miRNAs matched to the target genes were predicted. Figure 5 depicts the algorithm-predicted binding site of miRNA to the target genes. Luciferase reporter assay was employed to confirm the combination of miRNA and target genes. Following the manufacturer’s protocol, the position of the sequence region, containing the putative binding sequence of each miRNA, was inserted into a luciferase reporter vector pGLO-basic (Promega, Madison, WI, USA). The mutated sequence of the target gene was constructed into a pGLO-basic vector. The sequences constructed in the reporter vector and mutant vector were confirmed by sequence analysis. pRL-TK Renilla Luciferase Reporter vector (Promega, Madison, WI, USA) was used as an internal control vector. HEK 293 cells were seeded into 96-well plates with the confluent rate about 80% and co-transfected with either reported vector (0.01 mg/well each), miRNA mimic/mutated vector (10 nM) and internal control vector. After 48 h co-transfection, luciferase activities were measured by a microplate reader (Infinite M1000, TECAN, Männedorf, Swiss). Renin luciferase activity was defined as the standardization of firefly luciferase activity.
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6

Evaluating LTR16B2 Promoter Activity

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The long terminal repeat in ALK intron 19 at the ATI site (LTR16B2, chr2:29,446,649–29,447,062; 414 bp) was amplified using genomic DNA from patient MM-15 and 5′-GTCCTCATGGCTCAGCTTGT-3′ and 5′-AGCACTACACAGGCCACTTC-3′ primers. The PCR product (chr2:29,446, 444–29,447,174; 731 bp) was cloned into pGL4.14-firefly luciferase vector (Promega). To determine the promoter activity of LTR16B2, 105 cells were transfected in triplicates with 500 ng pGL4.14-LTR16B2 or vector alone; as internal control, 200 ng pRL-TK-Renilla luciferase reporter vector (Promega) was co-transfected. Luciferase activity was measured using Dual-Glo Luciferase Assay System (Promega) 48 h after transfection. Promoter activity was calculated by normalizing firefly luciferase activity to the control Renilla luciferase activity and compared between pGL4.14-LTR16B2 and vector alone. The luciferase reporter assays were independently performed three times, the results were combined, and the mean ± s.d. is shown in Extended Data Fig. 6g.
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7

Transcriptional Regulation Analysis via Luciferase Assay

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DNA fragments were amplified by PCR of human genomic DNA. The primer sequences are shown in Supplementary Table 1. All DNA fragments were inserted into the pGL3-LUC reporter vector (Promega, Madison, WI, USA). Mutants were generated using overlap-extension PCR. The SuperFect reagent (Qiagen, Hilden, Germany) was used to co-transfect 293T cells at a density of 2 × 105 with the pcDNA3 vectors (0, 10, 50, or 100 ng) with or without A/E cDNA and plated in 24-well plates. The pRL-TK renilla luciferase reporter vector (Promega, Madison, WI, USA) was used as a control. According to the manufacturer’s instructions, cells harvested 48 h after transfection were analyzed using the Dual-Luciferase® Reporter assay (Promega, Madison, WI, USA).
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8

Dual Luciferase Reporter Assay Protocol

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Cells with 70% confluence were co-transfected with 2.5 μg of each pMIR-REPORT construction and 0.05 μg of the pRL-TK Renilla Luciferase reporter vector (Promega, Charbonnières-les-Bains, France) by using the Lipofectamin 2000 reagent (Invitrogen, ThermoFisher Scientific). After 48 h of transfection, the reporter assays were carried out with the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Aliquots of cell lysates were transferred to a 96-well microplate and successively incubated with the Firefly Luciferase and Renilla Luciferase substrates. The luminescence signal was recorded with the FluoStar Optima Plate reader (BMG Labtech). Firefly luciferase values were first normalized to those of Renilla luciferase values, and then to luciferase expression of the empty pMIR-REPORT (assigned a value of 1) for each of the 3’UTR constructions (S1 Table). Assays were run in duplicates from at least three independent experiments.
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9

Validation of miR-145 Binding in KLF4 3'UTR

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By searching the Target Scan 6.2 database (http://www.targetscan.org/) to find algorithm-based binding sites of miR-145, we found a predicted binding site to be at positions 278–284 in the 3′-UTR of KLF4 mRNA. The sequence region 2161–2520, containing the putative binding sequence of miR-145, was inserted into a pMIR-REPORTTM Luciferase miRNA Expression Reporter Vector (Applied Biosystems) according the manufacturer's protocol. Moreover, we made another pMIR construct encompassing a mutated seed sequence for miR-145 (Wild type, GACTGGAA; mutant, GACGTCAA) by using a PrimeSTAR® Mutagenesis Basal Kit (TaKaRa). The mutation of the vector was confirmed by sequence analysis. The pRL-TK Renilla Luciferase Reporter vector (Promega, Madison, WI, USA) was used as an internal control vector. 253J B-V cells were seeded into 96-well plates at a concentration of 0.1 × 104 per well on the day before the transfection. The cells were co-transfected with either reporter vector (0.01 μg/well each) and 20 nM miR-145 or nonspecific non-coding siRNA (Dharmacon, Tokyo, Japan). Luciferase activities were measured at 24 h after co-transfection by using a Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol. Luciferase activities were reported as the firefly luciferase/Renilla luciferase ratio.
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10

Promoter Activity Assay in Stem Cells

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The promoter sequence of YB-1 target gene was cloned into pGL3 firefly luciferase reporter vector (Promega, USA) with sequence-specific primers (Table 1). Then, the recombinant pGL3 was co-transfected into melanoma stem cells or breast cancer stem cells with pRL-TK renilla luciferase reporter vector (Promega, USA). The activities of firefly luciferase of pGL3 and renilla luciferase of pRL-TK were determined following the dual-luciferase reporter assay protocol recommended by Promega. The cells were washed with PBS and then lysed with passive lysis buffer (Promega, USA). Subsequently, the cell lysate was subjected to the detection of firefly luciferase activity (M1), followed by the examination of renilla luciferase activity (M2). The promoter activity was calculated according to the following formula: Promoter Activity = M1/M2.
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