Quantitative Real-time PCR

Total RNA was extracted from cultured cells or primary cells using the TRIzol reagent (Life Technologies, Grand Island, NY), according to the manufacturer's instructions, and used to generate cDNA with a 5 × All-In-One RT MasterMix from Applied Biological Materials Inc (Richmond, BC, Canada). Realtime-PCR (qRT-PCR) was performed using SYBR Green kit (Qiagen, Hilden, Germany). For in vitro experiments, relative expression was calculated using the 2^-ΔΔCt method and normalized to the internal control gene GAPDH mRNA (human). For clinical data, relative expression was calculated using the 2^-ΔCt method and normalized against GAPDH mRNA values. The primers used are shown in Supplementary Primers.