Prl tk renilla control vector

Gastric organoids were dismantled by TrypLE Express. A total of 2.5 × 10⁵ cells were transfected with TOPFlash plasmid (Merck-Millipore) together with pRL-TK renilla control vector (Promega) using 4D-Nucleofector system (Lonza). Transfected cells were suspended in Matrigel and plated onto a 48-well multiple-well plate and incubated for 72 hours. The cells were then lysed, and luciferase activity was measured using Dual luciferase reporter assay kit (Promega).

SW1353 human chondrosarcoma cells (ATCC) were seeded at a density of 1.75 x 10⁴ cells per well in a 48-well plate 24 hours prior to transfection. MDA-MB-231 human adenocarcinoma cells (ATCC) were seeded at a density of 2.5 x 10⁴ cells per well in a 48-well plate 48 hours prior to transfection. SW1353 and MDA-MB-231 cells were co-transfected with 500 ng of the appropriate pGL3-promoter vector and 30 ng of pRL-TK Renilla control vector (Promega) using TurboFect transfection reagent (Thermo Scientific). After 24 hours, cells were lysed and luciferase and Renilla activity measured using the Dual Luciferase Assay System (Promega) according to the manufacturers’ protocol. For each construct, six wells were transfected and at least six independent experiments performed per cell line. The luciferase/renilla ratio of each construct was normalised to the empty pGL3-promoter vector, which was given a ratio of 1. The significance of expression differences were assessed using a Mann-Whitney U test.

DU145 cells were seeded at a concentration of 5 × 10⁴ cells per well in 6-well cell culture plates. After 24 h of incubation, cells were co-transfected with an miR-23a-3p mimic or negative control and vector containing the E-cad 3′UTR. The pRL-TK Renilla control vector (0.5 μg) (Promega) was also co-transfected as an internal control for transfection efficiency. GenMuteTM siRNA & DNA Transfection Reagent (SignaGen Laboratories, Ijamsville, MD) was used for this transfection process according to the manufacturer's instructions. Cells were harvested 48 h after transfection and analyzed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega).