RNA isolation and quantitative PCR were performed as described previously [
5]. Briefly, quantitative PCR was performed with SYBR-Green premix Ex Taq (Takara, Japan) and detected by a Real Time PCR System (Roche LightCycler 480 or Rotorgene 6000). β-Actin was used as an internal control gene. qPCR primers were designed using Primer Picking Program. The primer sequences for amplifying mouse cDNA fragments were as follows:
β-actin, forward, 5′-CGTCGACAACGGCTCCGGCATG-3′, reverse, 5′-CACCATCACACCCTGGTGCCTAGG-3′;
CX3CR1, forward, 5′-TTCCCATCTGCTCAGGACCTC-3′, reverse, 5′-CAGACCGAACGTGAAGACGA-3;
IBA1, forward, 5′-GGAGATTTCAAAAGCTGATGTGGA-3′, reverse, 5′-CCTCAGACGCTGGTTGTCTT′-3′;
iNOS, forward, 5′-TGCTTTGTGCGAAGTGTCAG-3′, reverse, 5′-CCCTTTGTGCTGGGAGTCAT-3′;
IL-1β, forward, 5′-AGGAGAACCAAGCAACGACA-3′, reverse, 5′-CTTGGGATCCACACTCTCCAG-3′;
IL-6, forward, 5′-GCCTTCTTGGGACTGATGCT-3′, reverse, 5′-TGCCATTGCACAACTCTTTTCT-3′;
IL-12β, forward, 5′-TGGTTTGCCATCGTTTTGCTG-3′, reverse, 5′-ACAGGTGAGGTTCACTGTTTCT-3′;
NeuN, forward, 5′-CCACCACTCTCTTGTCCGTT-3′, reverse, 5′-ATCAGCAGCGGCATAGACTC-3′;
NG2, forward, 5′-GGCTTGTGCTGTTCTCACA-3′, reverse, 5′-CACAGACTCTGGACAGACGG-3′;
Olig1, forward, 5′-CTCGCCCAGGTGTTTTGTTG-3′, forward, 5′-TATAAGCCTGCGCTACGACG-3′;
Pdgfrα, forward, 5′-TGGCAAAGAACAACCTCAG-3′, reverse, 5′-CGATAACCCTCCAGCGAAT-3′;
Pdgfrβ, forward, 5′-CATGTCTGAGACCCGGTACG-3′, reverse, 5′-CTCTGCAGGTAGACCAGGTG-3′;
PLP1, forward, 5′-AGCGGGTGTGTCATTGTTTG-3′, reverse, 5′-GCGCAGAGACTGCCTATACT-3′;
TNF-α, forward, 5′-ACGTCGTAGCAAACCACCAA-3′, reverse, 5′-ATAGCAAATCGGCTGACGGT-3′;
Csf1r, forward, 5′-CTCTTCCTCTGTTCCCTTTCAGG-3′, reverse, 5′-AGTTCTGTGAGGACGGGAAC-3′;
Sall1, forward, 5′-AACTAAGCCGAGGACCAAGC-3′, reverse, 5′-CTTCGGGGTCGGATTGGAAA-3′;
Trem2, forward, 5′-GGGTCACCTCTAGCCTACCA-3′, reverse, 5′-GTACCTCCGGGTCCAGTGA-3′;
Tmem119, forward, 5′-CACCCAGAGCTGGTTCCATA-3′, reverse, 5′-GTGACACAGAGTAGGCCACC-3′;
p2ry12, forward, 5′-ACCACCCCTGTTTTTCCAGT-3′, reverse, 5′-AGGCAGCCTTGAGTGTTTCTG-3′;
p2ry13, forward, 5′-TGGGTTGAGCTAGTAACTGCC-3′, reverse, 5′-TTGTCCCGAGCATCAGCTTT-3′;
MBP, 5′-CACCACTCTTGAACACCCCA-3′, reverse, 5′-TGCCCACGCTTCTCTTCTTT-3′; and
ALDH1L1, 5′-CCCGTCTTTGACCTTGGGTG-3′, reverse, 5′-GAACTTAAACACGGGCACGC-3′. The primer sequences for amplifying rat cDNA fragments were as follows: β-actin, forward, 5′-ACCCGCCACCAGTTCGCCAT-3′, reverse, 5′-CTAGGGCGGCCCACGATGGA-3′; CX3CR1, forward, 5′-GAGTGGCTGGCACTTCCTG-3′, reverse, 5′-CACCAGACCGAACGTGAAGA-3′; and
IL-1β, forward, 5′-TGCAGCTGGAGAGTGTGGATCC-3′, reverse, 5′-ACCAGTTGGGGAACTGTGCAGA-3′. The primer sequences for amplifying human cDNA fragments were as follows:
TGFβR2, forward, 5′-CACTGACAACAACGGTGCAGTC-3′, reverse, 5′-GCTTGGGGTCATGGCAAACTG-3′; and
CX3CR1, 5′-AGGTGGCCAAACACTGAGAC-3′, reverse, 5′-GTGAAGGCCTCTAGTCGCTG-3′. Following PCR amplification, a first derivative melting-curve analysis was performed to confirm the specificity of the PCR. The relative fold difference in mRNA between samples was calculated by comparing the threshold cycle (
Ct) at which product initially appeared above background according to 2
−(∆ Ct), where
∆Ct is the difference between the control group and a treatment group.