Caco-2 Cell Transfection and RNAi Analysis

Mammalian Caco2 cells were routinely cultured in complete growth media consisting of minimal essential medium (MEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mM l-glutamine, 200 µg/ml⁻¹ 0.1 mM nonessential amino acids, streptomycin, and 100 U·ml⁻¹ penicillin (37°C, 5% CO₂). Transient transfection of Caco2 cells by microporation was performed using a Neon transfection system according to the instructions of the manufacturer (Invitrogen). For RNA interference (RNAi) analysis, small interfering RNA (siRNA) from Qiagen and Dharmacon (Table 3) was transfected into Caco2 cells with Oligofectamine transfection reagent (Invitrogen) according to the instructions of the manufacturer. EFA6A and ARNO siRNA have been previously described (3 (link), 5 (link)). The transfection mixture was replaced after 24 h with complete growth medium, and cells were cultured for 72 h in total. RNA interference (RNAi) efficiency was determined by Express One-Step SYBR GreenER qRT-PCR according to the instructions of the manufacturer (Invitrogen) with actin used as a relative control in each case.