Total RNA was isolated with Trizol (Invitrogen, Carsbad, CA) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (PCR) was done with iQ SYBR Green Supermix kit (BioRad, Richmond, CA) by following the manufacturer’s instructions. Primer sequences for the analyzed genes are listed in Table 1. The PCR was carried out in a iCycler iQ at 95°C for 10 minutes, and 35 cycles of 95°C for 10 seconds, 55°C for 10 seconds, and 72°C for 10 seconds. The fold expression of each gene was calculated with the comparative cross-threshold method (Livak and Schmittgen, 2001) using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene.
