Real-time PCR
Total RNA was extracted using the TRIzol reagent (Molecular Research Center, Inc.) from NS20Y cells. DNase I-treated RNA (2 μg) was subjected to RT (Roche) using oligo(dT) primer. One-fortieth of this reaction was used for real-time PCR analysis using SYBR Green I dye chemistry. PCR product accumulation was monitored using a iCycler iQ Real-Time detection system (Bio-Rad). The mean cycle threshold value (Ct) from triplicate samples was used to calculate gene expression level. PCR products were normalized to levels of β-actin. Relative gene expression levels were determined as described in User's resource guide from Bio-Rad.