To construct reporter vectors bearing miRNA-target sites, the wild-type or mutant Gadd45β mRNA 3′UTR sequence was amplified by polymerase chain reaction (PCR) and cloned into pGL3-promoterconstruct (Promega), thus obtaining the wild-type or mutant Gadd45β 3′UTR firefly luciferase reporter gene. The cardiomyocytes were co-transfected with 80 ng wild-type or mutant Gadd45β 3′UTR firefly luciferase reporter gene, 40 ng Renilla luciferase reference plasmid pRL-TK and miR-206 per controls (final concentration, 20 nM). Forty-eight hours after transfection, the luciferase activity was measured by a dual-luciferase reporter gene assay system (Promega, USA). The final data were the ratio of firefly fluorescent value to Renilla fluorescence value.
Pgl3 promoter construct
Manufactured by Promega
The PGL3-promoter construct is a plasmid that contains a firefly luciferase gene coupled to a promoter sequence. This construct is commonly used as a tool to study gene expression and transcriptional regulation in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (2 mentions)
Dual luciferase reporter gene assay system, by Promega (1 mentions)
Prl tk renilla luciferase construct, by Promega (1 mentions)
Luciferase reporter system, by Promega (1 mentions)
X tremegene transfection reagent, by Roche (1 mentions)
Synergy h4 hybrid multiplate reader, by Agilent Technologies (1 mentions)
Prl tk, by Promega (1 mentions)
5 protocols using pgl3 promoter construct
1
Constructing miRNA-target Luciferase Reporters
2
Construct and Evaluate miRNA-Targeted Luciferase Reporter
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In order to construct reporter vector with miRNA-762 target site, wild-type or mutant UCP2 mRNA 3′UTR sequence was amplified by polymerase chain reaction (PCR) and cloned into pGL3-promoter construct (Promega). The wild-type or mutant UCP2 3′UTR firefly luciferase reporter gene was obtained. Myocytes were co-transfected with 80 ng wild-type or mutant UCP2 3′UTR firefly luciferase reporter gene, 40 ng Renilla luciferase reference plasmid PRL-TK, and miR-762 (final concentration, 20 nM) in each control. Luciferase activity was measured 48 h after transfection using the dual luciferase reporter assay system (Promega, USA). The final data is the ratio of firefly fluorescent value to Renilla fluorescence value.
3
Dual Luciferase Assay for FOXP1
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HEK293 cells were transfected with 50 ng of pGL3-promoter construct (Promega), 50 ng of pRL-TK renilla luciferase construct (Promega) and either empty pNTAP-B vector, TAP-tagged FOXP1-WT or TAP-tagged FOXP1-K670R. Cells were lysed 48 hr after transfection and both firefly and renilla luciferase activity quantified using the Dual Luciferase Reporter Assay System (Promega).
4
Luciferase Reporter Assay for Hypoxia Inducible Gene
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A 329 bp (hg19: chr8:128889122-128889450) insert was PCR amplified from genomic DNA and cloned into the pGL3 promoter construct (Promega) using KPNI and NHEI restriction sites. Primers are listed in Supplementary Table 5 . Transfections of plasmids (1 μg per well) were performed in Hela cells using X-treme gene transfection reagents (Roche). Cells were cultured in a 24-well plate, transfected with the plasmids at 30–50% cell density and stimulated with DMOG (1 mM) or vehicle for 16 h. Cells were collected 24 h after transfection. Luciferase activity was measured using a Luciferase reporter system (Promega). Cells were co-transfected with a plasmid expressing β-galactosidase and luciferase activity was normalized to the activity of β-galactosidase. All constructs were sequence verified.
5
Transcriptional Repression Activity Assay
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To evaluate transcriptional repression activity, we used a standard luciferase assay. 9 23 26 Mutations were introduced into the full length coding sequence of FOXP1 longest isoform (FOXP1a; obtained from Kazusa DNA Research Institute, Kisarazu, Japan) and subcloned into the mammalian expression vector pcDNA4His (Invitrogen). HEK293 cells were then cotransfected using Fugene 6 (Roche), in 24-well plates, with 400 ng of pcDNA4His without an insert or containing the wild-type FOXP1 (wt-FOXP1) or the mutant FOXP1 cDNA, along with 50 ng of pGL3-promoter construct (Promega) (in which the SV40 promoter drives a firefly luciferase reporter). To normalise for transfection efficiency and variation in cell number, cells were also cotransfected with 50 ng of a Renilla luciferase construct (pRL-TK; Promega) driven by the HSV-thymidine kinase promoter which is not affected by FOXP1. Cells were lysed 48 hours after transfection. Firefly and Renilla luciferase activities were quantified using the Synergy H4 Hybrid Multiplate Reader (BioTek). Protein expression was verified by western blotting (see below). Student's t-test was performed to evaluate statistical significance.
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