Itaq universal sybr green system

Total RNA was isolated from the mouse brain tissues and primary microglia using an RNeasy kit (Qiagen) and transcribed into cDNA using MultiScribe reverse transcriptase (Thermo Fisher Scientific). Expression levels of TNF, IL-6, IL-1β, CCL2 and CCL3-encoding mRNAs were determined by quantitative real-time PCR (iTaq Universal SYBR Green System, Bio-Rad) and normalized to the level of GAPDH. The following primers were used: mouse TNF-forward, 5′-GTCCCCAAAGGGATGAGAAGTT-3′; mouse TNF-reverse, 5′-CTCCTCCACTTGGTGGTTTG-3′; mouse IL-6-forward, 5′-TCCGGAGAGGAGACTTCACA-3′; mouse IL-6-reverse, 5′-TGCCATTGCACAACTCTTTTC-3′; mouse IL-1β-forward, 5′-TGCCACCTTTTGACAGTGATG-3′; mouse IL-1β-reverse, 5′-TGATGTGCTGCTGCGAGATT-3′; mouse CCL2-forward, 5′-CACTCACCTGCTGCTACTCA-3′; mouse CCL2-reverse, 5′-GCTTGGTGACAAAAACTACAGC-3′; mouse CCL3-forward, 5′-TCCCAGCCAGGTGTCATTTTC-3′; mouse CCL3-reverse, 5′-TCAGGCATTCAGTTCCAGGTC-3′; mouse GAPDH-forward, 5′-GAAGGTCGCTGTGAACGGA-3′; mouse GAPDH-reverse, 5′-GTTAGTGGGGTCTCGCTCCT-3′.