The binding sites between miR-143-3p and PCAT6 were predicted and analyzed using Starbase 2.0 (https://starbase.sysu.edu.cn/ ) and Lncbase v.2 (https://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=site%2Findex ). The binding sites between miR-143-3p and PDIA6 3'-untranslated regions (UTR) were predicted by TargetScan 7.1 (https://www.targetscan.org/vert_71/ ). The sequences of PCAT6 and PDIA6 3'UTR containing the potential wild-type (WT) or mutant (MUT) binding sites of miR-143-3p were synthesized by Tsingke Biological Technology Co., Ltd. and then inserted into the pGL3 vector (Guangzhou RiboBio Co., Ltd.) to construct the luciferase reporter. The T24T and EJ cells were seeded at a density of 1x104 into 96-well plates. The luciferase reporter (0.1 µg) and miR-143-3p mimics (40 nM) or mimic-NC (40 nM) were co-transfected into cells using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h of incubation at 37˚C, the transfected cells were harvested and assayed for luciferase activity using the dual-luciferase reporter assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.
Pgl3 vector
Manufactured by RiboBio
The PGL3 vector is a plasmid that is commonly used in molecular biology research. It contains a promoter sequence and a reporter gene, which can be used to study gene expression in various cell types. The core function of the PGL3 vector is to facilitate the measurement of transcriptional activity in experimental systems.
Automatically generated - may contain errors
2 protocols using pgl3 vector
1
miR-143-3p Targets PCAT6 and PDIA6 in Bladder Cancer
2
Validating miR-494 Interaction with SIRT3
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The bioinformatics algorithms and related databases were used to search for the potential targets for miR-494. SIRT 3 was confirmed to have an assumed binding site on miR-494. Wild-type (WT) and mutant (MUT) 3′-UTR fragments containing the assumed miR-494 binding site were amplified and inserted into the pGL3 vector (RiboBio). HEK 293 cells were seeded in a 6-well plate, grown in an incubator at 37 °C for 24 h with 5% CO2 and then co-transfected with 100 ng of pGL3 vector harboring MUT 3′-UTR or WT and 40 nM of miR-Scr or miR-494 mimic employing transfection reagent, lipofectamine 2000. The cells were harvested in 48 h to detect luciferase activity through a dual luciferase reporter assay kit (Promega, Madison, Wisconsin, USA).
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