Quantitative real-time PCR
Total RNA was isolated using RNAiso Plus reagent (TaKaRa) following the manufacturer’s instruction. First-strand cDNAs were synthesized from 500-1000 ng of total RNA with PrimeScript RT reagent Kit (TaKaRa) following the manufacturer’s instructions. Quantitative RT-PCR was performed using ABI StepOne Plus instrument and SYBR Green Supermix (Bio-Rad) and the primers are listed in Supplemental Table S1. The relative expression levels were calculated against the reference gene by the comparative CT method (Livak & Schmittgen, 2001).