We utilized a dual-luciferase reporter system (GeneCopoeia; # HmiT018728) in which the 3′-UTR of ZNF217 was fused to the end of Firefly luciferase. The construct also contains Renilla luciferase, which can be used as an internal control. Next, we mutated the vector to interfere with the binding of the miR-503 seed region (nucleotides 2–8) using a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent; #200521). Two A's were added in the miR-503 target site of ZNF217 to induce a bulge in the target site opposite of nucleotides 3 and 4 of miR-503's seed. MCF-7 cells were transiently transfected with the wild-type or mutant reporter with or cotransfected with miRIDIAN microRNA Human hsa-miR-503-5p mimic (Dharmacon #C-300841-05-0005) and with the vector. Luminescence was measured 48 h after transfection using the Luc-Pair Duo-Luciferase Assay (GeneCopoeia; # LPFR-P030) on a Promega GloMax Multi+ Detection System luminometer.
Luc pair duo luciferase assay
Manufactured by GeneCopoeia
The Luc-Pair Duo-Luciferase Assay is a laboratory equipment product that measures the activity of two different luciferase reporter genes simultaneously. The assay utilizes two distinct luciferase enzymes, each with a unique substrate, to provide a quantitative analysis of gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Glomax multi detection system luminometer, by Promega (1 mentions)
Mirvana mirna mimic, by Thermo Fisher Scientific (1 mentions)
X tremegene hp, by Roche (1 mentions)
4 protocols using luc pair duo luciferase assay
1
Quantifying miR-503 Binding to ZNF217
2
MiR-184 Target Validation in HeLa Cells
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HELA cells were co-transfected in a 96 well plate using GenMute™ siRNA Transfection Reagent with modified reporter vector (Genecopoeia pEZX-FR02) and 40pmol of miR-184 mimic (Life Technologies, mirVanaTM miRNA Mimics, MC10207) or miRNA mimic control (miR-scr, Life Technologies, 4464058). Forty-eight hours after transfection luciferase activities were measured using Luc-Pair Duo-Luciferase Assay from Genecopoeia, according to manufacturer’s instructions. All experiments were performed twice in triplicate. For each sample the Firefly luciferase activity was normalize to the Renilla luciferase activity used as a control to standardize for transfection efficiency.
3
miR-605 Regulation of CXCL5 Expression
4
Regulation of MECP2 Expression by miRNA
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HEK293 and Neuro-2a cells obtained from American Type Culture Collection (ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. For the reporter assay HEK293 cells were co-transfected with precursor miRNA or anti-miRNA plasmid (GeneCopoeia) and luciferase reporter plasmid containing the 3′UTR of Mecp2 using Lipofectamine 2000 (Life Technologies) for 48 h. The ∼8.5 kb 3′UTR sequence of Mecp2 was cloned downstream of the luciferase reporter gene as 4 constructs of ∼2 kb fragment each (GeneCopoeia). The fragment with the miRNA target sequence of interest (1–2210 bp or 6360–8591 bp) was co-transfected with corresponding miRNA. The Luc-Pair Duo-Luciferase assay (GeneCopoeia) was used to measure firefly and renilla luciferase according to the manufacturer’s instructions. Firefly luciferase measurements normalized to renilla was used as a transfection control. For monitoring changes in endogenous MeCP2, Neuro-2a cells were transfected with precursor miRNA or anti-miRNA plasmid (GeneCopoeia) using X-tremeGENE HP (Roche) for 48 h.
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