Negative nontarget control sirna pool

All siRNA pools with a set of four specific siRNAs for an individual candidate gene and a negative nontarget control siRNA pool were purchased from Dharmacon (Chicago, Illinois, USA). Reverse transfection of siRNA was performed in 96-well plates with a mixture of pancreatic cancer cells and 0.2 µl of lipofectamine RNAi-MAX reagent (Invitrogen, Carlsbad, California, USA), as well as 50 nmol/l of siRNA pools. At 24 h, cells were treated with gemcitabine (Eli Lilly, Indianapolis, Indiana, USA) at final concentrations of 0.001, 0.01, 0.1, 1, 10, 100, and 1000 µmol/l for 72 h. MTS cytotoxicity assays were performed using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit (Promega Corporation, Madison, Wisconsin, USA), followed by absorbance measurement at 490 nm in a Safire² microplate reader (Tecan AG, Männedorf, Switzerland).