2.10. Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated using TRIzol Reagent (Invitrogen), and first-strand cDNA was synthesized from 1 µg total RNA using a QuantiTect Rereverse Transcription Kit (Qiagen, Valencia, CA, USA). qRT-PCR was performed using a QuantiFast SYBR Green PCR master mix (Qiagen) with an CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). The data were analyzed by comparative Ct quantification, and the value for each sample was normalized to the value for the housekeepingGAPDH gene. Primer sets for T-bet, GATA-3, RORγt, IFN-γ, IL-4, IL-17A, and GAPDH were obtained from Qiagen. The amplification program consisted of 1 cycle of 95°C for 5 min followed by 25 cycles of 96°C for 10 sec, 56°C for 20 sec, and 72°C for 20 sec.