Prl tk renilla plasmid

TargetScan (version 6.0; http://www.targetscan.org/vert_60/), microcosm (version 1.1; http://tools4mirs.org/software/mirna_databases/microcosm-targets/) and miRanda (version August 2010; http://www.microrna.org/microrna/home.do) were used to predict miRNAs that could potentially target PDGFRA and identify possible binding regions. The fragment of the human PDGFRA with [wild type (wt)] or without [mutant (mut)] the miR-342-3p binding site at the 3′-untranslated region (3′-UTR) was cloned and inserted into the pGL3-basic luciferase report plasmid (Promega Corporation) to generate the luciferase reporter vectors, PDGFRA 3′-UTR-wt and PDGFRA 3′-UTR-mut. 293T cells (American Type Culture Collection) were treated in 96-well plates at 5,000 cells per well and incubated for 24 h at 37°C with 5% CO₂ prior to transfection. Then, miR-342-3p mimics or miR-negative control was transfected into 293T cells using Lipofectamine 2000 with 100 ng of PDGFRA 3′-UTR-wt or PDGFRA 3′-UTR-mut, or 10 ng of pRL-TK Renilla plasmid (Promega Corporation). Following incubation for 48 h, the luciferase activities were determined with a Dual Luciferase Reporter System (Promega Corporation); Renilla luciferase was used for normalization.

Cells (2×10⁴/well) were seeded in triplicate in 48-well plates and allowed to settle for 24 h. Next, 100 ng of the luciferase reporter plasmids or the control plasmid, both with 1 ng of pRL-TK Renilla plasmid (Promega Corporation), were transfected into cells using the Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific), according to the manufacturer's recommendation. Luciferase and Renilla signals were assessed 24 h after transfection using a Dual Luciferase Reporter assay kit (Promega Corporation), according to the manufacturer's instructions.