The effect of 12-HHT/BLT2 receptor axis on the global gene expression after calcium switch in MDCK cells

Examination of global gene expression in BLT2 overexpressing MDCK (MDCK-BLT2) cells and Mock-MDCK cells before and after calcium switch. After MDCK cells reached confluence, culture medium (DMEM (WAKO) containing 10% FCS) was replaced to calcium-free medium SMEM (Gibco) and incubated for 2 h or 18 h. After incubation for 18 h, SMEM was changed to high-calcium medium DMEM (WAKO) with or without 200 nM 12-HHT, and the cells were incubated for 2 h. Total RNA was prepared from MDCK cells using TRIzol Reagent (Life Technology). Total RNA from triplicate samples was mixed to reduce the difference among the samples, and subsequently used for labeling. cDNA was generated and labeled with The Ambion WT Expression Kit and GeneChip WT Terminal Labeling Kit (Affymetrix) according to the manufacturer's protocols. The labeled samples were subjected to hybridization with GeneChip Canine Gene 1.0 ST Array (Affymetrix) and GeneChip Hybridization, Wash, and Stain Kit (Affymetrix). The signals were scanned using the GeneChip Scanner 3000 7G, and the data were analyzed using Affymetrix Expression Console Software.