2.10. Determination of Gene Expression

Cells and tissues were collected to synthesize cDNA, and mRNA expression was measured using qPCR (quantitative real-time PCR). Total RNA isolation and cDNA synthesis were performed using the RNeasy mini kit and ImProm-II Reverse Transcription System, respectively. qPCR was performed using the 7500 Fast Real-Time PCR system (Applied Biosystems, Singapore) and Fast SYBR^® Green Master Mix. All reaction experiments were independently repeated three times, and the data were analyzed using the comparison cycle threshold (Ct) method [26]. PCR primers for IL-4 ({"type":"entrez-nucleotide","attrs":{"text":"NM_021283","term_id":"226874825","term_text":"NM_021283"}}NM_021283) were 5′-ACC TTG CTG TCA CCC TGT TC-3′ (forward), 5′-TTG TGA GCG TGG ACTCAT TC-3′ (reverse); TNFα ({"type":"entrez-nucleotide","attrs":{"text":"NM_013693","term_id":"518831586","term_text":"NM_013693"}}NM_013693) were 5′-AGC CCC CAG TCT GTA TCC TT-3′ (forward) and 5′-CTC CCT TTG CAG AAC TCA GG-3′ (reverse); IL-1β ({"type":"entrez-nucleotide","attrs":{"text":"NM_008361","term_id":"921274059","term_text":"NM_008361"}}NM_008361) were 5′-CAA CCA ACA AGT GAT ATT CTC CAT G-3′ (forward) and 5′-GAT CCA CAC TCT CCA GCT GCA-3′ (reverse); IL-25 ({"type":"entrez-nucleotide","attrs":{"text":"NM_080729","term_id":"298676501","term_text":"NM_080729"}}NM_080729) were 5′-CAG CAA AGA GCA AGA ACC-3′ (forward) and 5′-CCC TGT CCA ACT CAT AGC-3′ (reverse); IL-33 ({"type":"entrez-nucleotide","attrs":{"text":"NM_133775","term_id":"1341395583","term_text":"NM_133775"}}NM_133775) were 5′-CAA TCA GGC GAC GGT GTG GAT GG-3′ (forward) and 5′-TCC GGA GGC GAG ACG TCA CC -3′(reverse); for β-actin ({"type":"entrez-nucleotide","attrs":{"text":"NM_007393","term_id":"930945786","term_text":"NM_007393"}}NM_007393) were 5′-TCA TCA CCA TCG GCA ACG-3′ (forward), 5′-TTC CT GAT GTC CAC GTC GC-3′ (reverse) [27]. The mRNA expression was normalized with β-actin and calculated based on the ratio to 100% of the phorbol 12-myristate 13-acetate (PMA)/ionomycin-treated group, the oxazolone-treated group or DNCB-treated group.