Transfection and the Dual‐Glo™ Luciferase Assay System (Promega) were performed as described before.13 As an internal control, pRL‐CMV plasmids (Promega) were cotransfected to normalize the transfection efficiency in the different transfection samples. Renilla luciferase activities from the pRL‐CMV plasmid were not affected by the cotransfection with K‐RTA. Triplicated transfection was performed in each reporter assay. The ratio between firefly and Renilla luciferase activities from the same sample was calculated to obtain the normalized firefly luciferase activity. Average and standard deviations (SD) were calculated, and reproducibility was confirmed.
Prl cmv plasmid
Manufactured by Promega
Sourced in United States
The PRL-CMV plasmid is a laboratory tool used for gene expression studies. It contains a promoter sequence that drives the expression of a target gene in transfected cells. The plasmid can be used to introduce and express genes of interest in various cell lines for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (7 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter assay kit, by Promega (3 mentions)
Pgl3 control plasmid, by Promega (2 mentions)
Pgl3 basic vector, by Promega (2 mentions)
Glomax multi detection system, by Promega (2 mentions)
Transit 2020 transfection reagent, by Mirus Bio (2 mentions)
Veritas microplate luminometer, by Promega (2 mentions)
Dual luciferase kit, by Promega (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Pgl3 vector, by Promega (2 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Lipofectamine 2000, by Promega (1 mentions)
Pgl3 construct, by Promega (1 mentions)
Pgl3 control vector, by Promega (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
25 protocols using prl cmv plasmid
1
Dual-Glo Luciferase Assay for Transfection
2
Validation of miR-130a-3p targeting BACH2
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After synthesizing the wild-type and mutant-type fragments of BACH2, the two fragments were amplified by PCR. The PCR products were then inserted into T4 vector with polyA and transformed in Escherichia coli DH5α. The monoclones were used for PCR confirmation. Thereafter, the successfully constructed plasmids and pGL-3 control plasmids (Promega, Madison, WI, U.S.A.) were digested with Xbal restriction enzymes to generate the target fragments, which were then ligated into pGL-3 control vector, respectively. Finally, PCR electrophoresis and DNA sequencing were used to confirm the pGL-3-BACH2 3′-UTR recombinant vector.
The constructed pGL-3-BACH2 3′-UTR plasmids and pRL-CMV plasmids (Promega, Madison, WI, U.S.A.) were transferred into CNE cells, respectively, using Lipofectamine™ 2000. Cells were divided into six groups: pGL-null + mimics negative control, pGL-null + miR-130a-3p mimics, pGL-3-BACH2-WT + mimics negative control, pGL-3-BACH2-WT + miR-130a-3p mimics, pGL-3-BACH2-MUT + mimics negative control, pGL-3- BACH2-MUT + miR-130a-3p mimics. A luciferase assay kit was used, in accordance with the manufacturer’s instructions, to measure the luciferase activity of the cells.
The constructed pGL-3-BACH2 3′-UTR plasmids and pRL-CMV plasmids (Promega, Madison, WI, U.S.A.) were transferred into CNE cells, respectively, using Lipofectamine™ 2000. Cells were divided into six groups: pGL-null + mimics negative control, pGL-null + miR-130a-3p mimics, pGL-3-BACH2-WT + mimics negative control, pGL-3-BACH2-WT + miR-130a-3p mimics, pGL-3-BACH2-MUT + mimics negative control, pGL-3- BACH2-MUT + miR-130a-3p mimics. A luciferase assay kit was used, in accordance with the manufacturer’s instructions, to measure the luciferase activity of the cells.
3
TP63 3'UTR Luciferase Assay
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The full fragment of TP63 3'UTR containing the potential binding site of miR-140 was amplified using PCR and the binding site was mutated using a Quik-Change Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA, USA). Subsequently, wild-type and mutant TP63 3'UTR was inserted into a pGL3-control vector (Promega, Madison, WI) immediately downstream of the luciferase reporter gene to generate different luciferase constructs. The cells were plated into 96-well plates at a final density of 1×10 5 cells/well and cultured for 12 hours, followed by co-transfection with 0.5 mg pGL3 constructs, 0.07 mg pRL-CMV plasmids (Promega, Madison, WI), and a miR-140 mimic or inhibitor. The transfection was carried out using Lipofectamine 3000 (Invitrogen, CA, US). At 48 hours post-transfection, a Dual Luciferase Reporter Assay System (Promega, Madison, WI) was utilized to measure the activity of firefly and renilla luciferase. The expression of different constructs was calculated by normalizing the firefly signal to the activity of renilla luciferase. Each test was repeated three times.
4
Lipofectamine-Mediated Transfection and Luciferase Assay
5
Investigating miR-199a Regulation in PC-3 Cells
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PC-3 cells were cultured in 24-well plates and transfected with 0.4 μg of the reporter constructs using Lipofectamine 2000 (11668019; Invitrogen, Carlsbad, CA). The pRL-CMV plasmid (E2261; Promega, Madison, WI) containing the Renilla luciferase gene (0.02 μg) was cotransfected as an internal control. Cells were infected with AD-miR-199a or AD-Control (MOI 100) at 4 h after transfection and collected 24 h later. Firefly and Renilla luciferase activities were assessed using a Luminometer TD-20/20 (Turner Design, Sunnyvale, CA).
6
Dual-Luciferase Promoter Activity Assay
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The promoter regions of HCCR-1 (1,632 bp) and p21 (934 bp) were inserted into pGL3-Basic Vector (Promega Corp.) using a recombinant cloning technology (GenScript). Cells with 70% confluence were cotransfected with 1 µg of promoter/pGL3 basic chimerical plasmid (expressing firefly luciferase) and 0.02 µg of pRL-CMV plasmid (expressing Renilla luciferase) (Promega Corp.), following the instructions of FuGENE HD (Promega Corp.). Promoter activities were detected following the protocol of Dual-Luciferase® Reporter Assay System (Promega Corp.) on a Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek, Winooski, VT, USA). Firefly luciferase values were standardized to Renilla values.
7
Investigating NF-κB and iNOS Regulation
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To investigate NF-κB transcriptional activity, RAW 264.7 were plated in 48-well polystyrene plates (1 × 105 cells per well) and transfected with 1 μg of the p6kB-Luc luciferase reporter construct (kindly provided by Dr. Patrick Baeuerle, Munich University) in the presence of LIPOFECTAMINE 2000 reagent (Invitrogen, Carlsbad, CA, USA). pTK-3XNS luciferase reporter construct was used to measure iNOS promoter activity, provided by Dr. David Geller (University of Pittsburgh, Pennsylvania, EUA). Luciferase activity was normalized using 40 ng of pRL-CMV plasmid (Promega Corp., Madison, WI, USA). Transfected cells were infected with either WT, Δlpg1 or Δlpg1+LPG1 parasites at a 10:1 MOI. After 24 h of infection, cells were washed with PBS, lysed according to the Dual Luciferase System protocol (Promega Corp.), and analyzed in a GloMax®-Multi detection system (Promega Corp.). Positive controls consisting of cells stimulated with 1 μg/mL of LPS (Sigma-Aldrich) were used to induce the activation of iNOS gene expression.
8
Luciferase Assay for Gene Expression
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Recombinant plasmids or an empty plasmid encoding the firefly luciferase reporter were transiently transfected into SW1990-sh-NORAD and SW1990-sh-NC cells using Lipofectamine 2000 (Invitrogen, USA) The renilla luciferase reporter pRL-CMV plasmid (Promega) was also transfected into these cells as an internal control. Each group above was transfected with miR-125a-3p mimic or a negative control. Luciferase activities in the indicated cells were measured using the Dual-Luciferase Reporter Assay System (Promega, USA) 48 h after transfection according to the manufacturer’s instructions.
9
NF-κB and IL-6 Promoter Luciferase Assays
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Luciferase assays were performed as described previously.21 (link) Briefly, cells grown in 6-well plates were transfected with an NF-κB firefly luciferase reporter plasmid 3X-κB-Luc (a gift from Dr. Hiroyuki Inuzuka) and a firefly luciferase reporter containing the IL-6 promoter (a gift of Dr. Hiroyuki Inuzuka) along with a pRL-CMV plasmid (Promega, Madison, WI, USA). After 24 h, cells were split into 96-well plates. After stimulation with aldosterone, firefly luciferase activity was measured using the Dual Luciferase Assay System and normalized to renilla luciferase activity according to manufacturer's instructions (Promega, Madison, WI, USA).
10
AP-1 and NF-κB Activation Assays
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The pGL-AP-1 plasmid containing six consensus AP-1 binding sites was co-transfected with the pRL-CMV plasmid (Promega) into cells using TransIT 2020 transfection reagent (Mirus Bio, MIR5400). A pRL-CMV plasmid expressed Renilla protein and was used as an internal transfection control. Twenty-four hours after transfection, cells were split and treated with tamoxifen for 24 hr to induce transformation. After 3 days, firefly and Renilla luciferase activities were determined by Dual-luciferase Reporter Assay kit (Promega, E1910). To evaluate NF-κB activity, pGL4.32 (luc2P/NF-κB-RE/Hygro) (E8491, Promega) and pRL-CMV were co-transfected into cells and the same protocol followed.
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