Real-time PCR Gene Expression Analysis

Real-time PCR was performed as described previously [22 (link)]. Briefly, the brain tissues were homogenized in Trizol reagent (Life Technologies, Carlsbad, CA) and total RNA was extracted following the manufacturer's instructions. 1 µg total RNA from each sample was reverse transcribed into cDNA using a RETROscript® reverse transcription kit (Life Technologies, Carlsbad, CA). Quantitative PCR was performed with ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) using SYBR® Green PCR Master Mix (Life Technologies, Carlsbad, CA). Conditions for amplification were 1 cycle of 94℃ for 5 min followed by 40 cycles of 94℃ for 30 s, 58℃ for 30 s, and 72℃ for 30 s. The primer sequences used in this study were as follows: CD68 F: 5'-CTGTTGCGGAAATA CAAGCA-3', R: 5'-GGCAGCAAGAGAGATTGGTC-3'; IL-1β F: 5'-GACCTGTTCTTTG AGGCTGACA-3', R: 5'-CTCATCTGGACAGCCCAAGTC-3'; TNF-α F: 5'-CATCTTCT CAAAACTCGAGTGACAA-3', R: 5'-TGGGAGTAGATAAGGTACAGCCC-3'; GAPDH F: 5'-ATGACTCTACCCACGGCAAG-3', R: 5'-CTGGAAGATGGTGATGGGTT-3'.