qRT-PCR Analysis of Gene Expression

Total RNA from PMs was isolated and reverse transcribed into cDNA as described above. qRT-PCR was conducted using the SYBR Green PCR premix (TaKaRa, Dalian, China) and run on a qRT-PCR instrument (iQ5 Bio-Rad, Hercules, CA, USA) as described previously [41 (link)]. The reaction conditions were as follows: stage 1, 95 °C for 30 s; stage 2, 40 cycles of 95 °C for 5 s and 60 °C for 30 s; and stage 3, melting curve analysis. The relative expression of the target genes was determined by the comparative quantification cycle (Cq) normalized against the housekeeping gene (GAPDH) using the 2^−ΔΔCq method [29 (link), 42 (link)]. The sequences of all primers used in this analysis are shown in Table 1.Table 1

Sequences of the qRT-PCR primers

Gene	GenBank ID	Forward primer	Reverse primer
iNOS	NM_010927.3	TTCACCCAGTTGTGCATCGACCTA	TCCATGGTCACCTCCAACACAAGA
TNF-α	NM_013693.3	AAGCCTGTAGCCCACGTCGTA	AGGTACAACCCATCGGCTGG
IL-10	NM_010548.2	GCCAGAGCCACATGCTCCTA	GATAAGGCTTGGCAACCCAAGTAA
Ym1	NM_009892.3	TCTCTACTCCTCAGAACCGTCAGA	GATGTTTGTCCTTAGGAGGGCTTC
Fizz1	NM_181596.4	TACTTGCAACTGCCTGTGCTTACT	TATCAAAGCTGGGTTCTCCACCTC
Arg1	NM_007482.3	CTCCAAGCCAAAGTCCTTAGAG	AGGAGCTGTCATTAGGGACATC
GAPDH	NM_001289726.1	CATGGCCTTCCGTGTTCCTA	CCTGCTTCACCACCTTCTTGAT