RNA extraction and real-time PCR
The spleen was taken from the sacrificed mice for RNA extraction using
TRIzol™ Reagent (ThermoFisher, 15596026). RNA was reverse transcribed
into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo
Fisher Scientific, K1622). The concentration and integrity of RNA and
cDNA were measured using a NanoDrop (Thermo Fisher, 2000). The mRNA
expression values of the genes were normalized to the mRNA expression ofactin . The results were calculated using the comparative cycle
threshold (ΔΔCt) method. The primers used were as follows: IL-4(F):
GGTCTCAACCCCCAGCTAGT; IL-4(R): GCCGATGATCTCTCTCAAGTGAT; IL-5(F):
CTCTGTTGACAAGCAATGAGACG; IL-5(R): TCTTCAGTATGTCTAGCCCCTG; IL-6(F):
TAGTCCTTCCTACCCCAATTTCC; IL-6(R): TTGGTCCTTAGCCACTCCTTC; IL-10(F):
GCTCTTACTGACTGGCATGAG; IL-10(R): CGCAGCTCTAGGAGCATGTG; IL-13(F):
CCTGGCTCTTGCTTGCCTT; IL-13(R): GGTCTTGTGTGATGTTGCTCA; IL-17(F):
TTTAACTCCCTTGGCGCAAAA; IL-17(R): CTTTCCCTCCGCATTGACAC; ICAM-1(F):
GTGATGCTCAGGTATCCATCCA; ICAM-1(R): CACAGTTCTCAAAGCACAGCG; IFN-γ(F):
ATGAACGCTACACACTGCATC; IFN-γ(R): CCATCCTTTTGCCAGTTCCTC; CCL-11(F):
GAATCACCAACAACAGATGCAC; CCL-11(R): ATCCTGGACCCACTTCTTCTT; TGF-β1(F):
TCTGCATTGCACTTATGCTGA; TGF-β1(R): AAAGGGCGATCTAGTGATGGA; TGF-β2(F):
CCGCTGCATATCGTCCTGTG; TGF-β1(R): AGTGGATGGATGGTCCTATTACA; IKK(F):
ACAGCCAGGAGATGGTACG; IKK(R): CAGGGTGACTGAGTCGAGAC; IKB(F):
GCGGATGCCGATGAATGGT; IKB(R): TGACGTAGCCAAAGACTAAGGG; JUN(F):
CCTTCTACGACGATGCCCTC; JUN(R): GGTTCAAGGTCATGCTCTGTTT; JNK(F):
CAGACTGTACCCTCAAGATCCT; JNK(R): ATGCACCCGACAGACCAGA; ERK(F):
GGTTGTTCCCAAATGCTGACT; ERK(R): CAACTTCAATCCTCTTGTGAGGG; FOS(F):
CGGGTTTCAACGCCGACTA; FOS(R): TTGGCACTAGAGACGGACAGA.