Fragments bound to miR-376c-3p in linc01278 and DNM3 3ʹ UTR were separately mutated using the QuikChange site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, USA). The chemically synthesized wild-type and mutant linc01278 or DNM3 3ʹ UTR fragments containing the miR-376c-3p binding site were separately cloned into the psiCHECK2 vector. The psiCHECK2 recombinant plasmid and miR-376c-3p mimic or miR-NC were co-transfected into BCPAP cells using Lipofectamine 2000. 48 h after transfection, firefly and Renilla luciferase activities in cell lysates were determined by the Dual Luciferase Reporting Assay System (Promega Corporation). Relative luciferase activity was normalized with Renilla luciferase activity.
Dual luciferase reporting assay system
Manufactured by Promega
Sourced in United States
The Dual-luciferase reporting assay system is a laboratory equipment used to measure and compare the activities of two different luciferase reporter enzymes simultaneously in a single sample. It provides a quantitative analysis of gene expression and regulatory elements.
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Lab products found in correlation
4 protocols using dual luciferase reporting assay system
1
Dual Luciferase Assay for miR-376c-3p Targets
2
Promoter Activity Assay for H. zea
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One hundred uL of H. zea fatbody cells were seeded onto wells of 96-well plates (5 × 105 cells/well). After 30 min, the seeded cells were transiently co-transfected with either of the 6 wildtype (pGL3-CCE001j promoter, pGL3-CCE001b promoter, pGL3-CCE001f promoter, pGL3-CYP6AE19 promoter, pGL3-UGT40F2 promoter, pGL3-ALDH1A1L promoter) and 4 deletion promoter-pGL3 constructs (del-Motif 1, del-Motif 2, del-ARE1 and del-ARE2) (0.1 µg/well) and the internal renilla luciferase control reporter plasmid PHRL-TK (Promega, 0.01 µg/well). There were three technical replicates in both the experimental group and the control group. Six hours after transfection, the final concentration of 18.5 μM flavone (induced) or equivalent volume of methanol (control) was added. After 48 h, the cells were harvested and the luciferase activity of kidney and firefly was measured using the dual luciferase reporting assay system (Promega, Madison, WI, USA) on a TD-20/20 single-tube luminescence instrument designed by Turner (Turner Biosystems, Sunnyvale, CA, USA). The relative firefly luciferase activity normalized against the renilla luciferase activity was calculated as an indicator of the basal or flavone-inducible promoter activity of each construct. At least 3 repeated measurements were conducted for each independent transfection.
3
Regulation of SNAI2 by lncRNA AC005224.4 and miR-140-3p
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The targeted relationship between lncRNA AC005224.4 and miR-140-3p was predicted using ENCORI database (http://starbase.sysu.edu.cn ). The targeted relationship between SNAI2 and miR-140-3p was predicted using TargetScan v7.2 database (http://www.targetscan.org/vert_72/ ). PmirGLO, PmirGLO-AC005224.4-wt/PmirGLO-AC005224.4-mut, and PmirGLO-SNAI2-wt/PmirGLO-SNAI2-mut were commercially obtained from Youbio (Changsha, China), and these were then co-transfected with miR-140-3p mimics/inhibitor or mimics/inhibitor control into 293 T cells using Lipofectamine-mediated gene transfer. After transfection for 48 h, cells were harvested and the luciferase activity was determined according to the protocol of dual-luciferase reporting assay system (Promega, Madison, WI, USA).
4
Luciferase Assay for LINC00242-miR-1-3p-G6PD Interaction
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The targeted correlation between LINC00242 and miR-1-3p was predicted using ENCORI database (http://starbase.sysu.edu.cn ). Then, targeted relationship between miR-1-3p and G6PD was predicted using TargetScan v7.2 database (http://www.targetscan.org/vert_72/ ). PmirGLO, pmirGLO-LINC00242-wt or pmirGLO-LINC00242-mut, pmirGLO-G6PD-wt or pmirGLO-G6PD-mut were commercially acquired from Youbio (Changsha, China), and then was co-transfected with miR-1-3p mimics/inhibitor or mimics/inhibitor control into 293 T cells by Lipofectamine-mediated gene transfer. Cells were collected 48 h after transfection and luciferase activity was determined according to the protocol of dual-luciferase reporting assay system (Promega, Madison, WI, USA).
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