RNA Extraction, cDNA Synthesis, and RT-PCR Analysis

RNA was extracted from cell culture lysates or liver tissue using a RNeasy Mini Kit (Qiagen, Germantown, MD) according to the manufacturer’s instructions. Synthesis of cDNA was accomplished using a Bio-Rad iScript™ cDNA Synthesis Kit (Hercules, CA). RT-PCR was performed as previously described [23 (link)] using commercially available primers designed against mouse TGFβ1, CCL2, CX3CL1, interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNFα), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SABiosciences, Frederick, MD). A ΔΔCT analysis was performed using vehicle-treated tissue or untreated primary neurons as controls for subsequent experiments [24 (link), 25 (link)].