LPS-induced gene expression analysis

To prepare total RNA, Sepasol (Nacalai Tesque) was added to BV-2 cells and the right striatum of each mouse was injected with LPS, and RNA fractions were extracted by following protocol. Complementary DNA (cDNA) was prepared by reverse transcriptional reaction using ReverTraAce and an oligo (dT)₂₀ primer (Toyobo Life Science, Inc., Tokyo, Japan). The expression of interested gene expression was analyzed by quantitative real-time PCR using an ABI 7300 thermal cycler and Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) as previously described [32 (link)]. The PCR primer sequences were as follows: iNOS 5′-TCTGCGCCTTTGCTCATGAC-3′(Upstream) and 5′-TAAAGGCTCCGGGCTC-3′ (downstream); IL-6 5′-CCACTTCACAAGTCGGAGGC-3′ (Upstream) and 5′-GGAGAGCATTGGAAATTGGGGT-3′ (downstream); TNFα 5′-TACTGAACTTCGGGGTGATCGG-3′ (Upstream) and 5′-CAGCCTTGTCCCTTGAAGAGAA-3′ (downstream); CCL2 5′-TGAGGTGGTTGTGGAAAAGG-3′ (Upstream) and 5′-CCTGCTGTTCACAGTTGCC-3′ (downstream); CXCL1 5′-GCCTATCGCCAATGAGCTG-3′ (Upstream) and 5′-TGGGGACACCTTTTAGCATC-3′ (downstream); COX-2 5′-AGAAGGAAATGGCTGCAGAA-3′ (Upstream) and 5′-GCTCGGCTTCCAGTATTGAG-3′ (downstream); GAPDH 5′-ACTCCACTCACGGCAAATTC-3′ (Upstream) and 5′-CCTTCCACAATGCCAAAGTT-3′ (downstream). The expression of GAPDH mRNA was utilized as an internal control.