Quantitative RT-PCR Analysis of Key Transcription Factors

Total RNA was extracted from PBMCs at DPC 0 and TBLN at DPC 6 using TRIzol reagent (Invitrogen, CA, USA) as per the manufacturer’s instructions. NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scientific, MA, USA) was used to determine the concentration and purity of RNA. cDNA was prepared from 1 μg of total RNA using the QuantiTect Reverse Transcription Kit (AIAGEN). Primers of housekeeping gene (β actin) and target genes (T-bet and GATA-3) used in this experiment were described previously (36 (link)). The mRNA expression was analyzed by 7500 Real-Time qPCR system (Applied Biosystems, CA, USA) using the qScript™ One-Step SYBR Green qRT-PCR kit, Low ROX™ (Quantabio, MA, USA). The target gene expression level was normalized with housekeeping gene levels, and the fold change was determined by comparative 2^−ΔΔCT method (37 (link)).