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.