Dharmafect duo transfection reagent

CHO-K1 cells were obtained from the Korean Cell Line Bank (KCLB No. 10061) and cultured in Ham’s F-12 medium containing 10% fetal bovine serum and 1 × Antibiotic-Antimycotic (Life Technologies). Human mirVANA miRNA mimics were obtained from Life Technologies. These included hsa-miR-96 (assay ID: MC10422), hsa-miR-182 (assay ID: MC12369), hsa-miR-183 (assay ID: 12830), and negative control #1 (cat# 4464058). Cells were plated at a density of 2.5 × 10³ in 48-well plates on day 0. On day 2, pGLO plasmids (0.3 μg/well) and mirVANA miRNA mimics (0, 1, 3, 10, and 20 nM) were transfected into cells using DharmaFECT-Duo Transfection Reagent (GE Healthcare). Cell lysates were prepared in 1× lysis buffer (Promega). Firefly luciferase and Renilla luciferase activities were measured using a Dual-luciferase System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity.

Rat LA7 cells (obtained directly from ATCC, catalog #: CRL-2283), which were originally derived from a DMBA-induced mammary tumor in a Sprague Dawley rat, and human MCF7 cells (obtained directly from ATCC, catalog #: HTB-22) were used in in vitro experiments. MicroRNA-200a inhibition was performed using 5nM miR-200a-targeting and non-targeting control power inhibitors (Exiqon), transfected in tandem with a synthetic miR-200a target RenSP luciferase reporter plasmid (Switchgear Genomics, Carlsbad, CA) at a ratio of 1:5 (ng:nL) with Dharmafect Duo transfection reagent (GE Healthcare, Pittsburgh, PA). Twenty-four hours after transfection, cells were seeded into two separate 96-well plates, one for luciferase readout to verify miRNA inhibition and one for proliferation analysis. Luciferase and proliferation analyses were done 24 hours after reseeding, for a total of 48 hours after miRNA inhibition. RenSP luciferase signal was analyzed using LightSwitch Assay Reagent (Switchgear Genomics) according to manufacturer protocol. Cellular proliferation was measured using a BrdU ELISA kit (Roche, Basel, Switzerland) following manufacturer protocol.

The 3′UTR containing predicted miR-29 binding sites, both wild type and mutant, for ATG9A and TFEB were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, #E1330) downstream of the firefly luciferase open reading frame. 5×10³ pancreatic cancer cells per well (Panc-1 or MIA PaCa-2) were plated in 96 well plates and grown at 37°C for 24 hours. Cells were then co-transfected for 24hrs with 10nM mimic control or miR-29a mimic and 100ng of pmirGLO Dual-Luciferase miRNA Target Expression Vector containing each respective 3′UTR binding site using DharmaFECT Duo Transfection Reagent (GE, T-2010-02). Cells were transfected for 24 hours, and luciferase levels were measured 24 hours post-transfection using Dual-Glo® Luciferase Assay System (Promega, #E2920). Firefly luciferase luminescence was normalized to renilla luciferase activity for each transfected well.