Microglia RT-PCR analysis

mRNA was isolated from primary microglia (3.1 × 10⁴ cells/cm²) after stimulation with chromogranin-A (CGA) (synthetic human CGA286-301, Peptide Institute, Osaka, Japan), or after pre-treatment with Naturido according to previous described methods [26]. Total RNA was extracted with RNAiso Plus (TaKaRa, Japan) according to the manufacturer’s instructions. A total of 800 ng of extracted RNA was reverse transcribed into cDNA using a QuantiTect Reverse Transcription Kit (Qiagen, Japan). After an initial denaturation step at 95°C for 5 min, temperature cycling was initiated. Each cycle consisted of denaturation at 95°C for 5 s, annealing at 60°C for 10 s, and elongation for 30 s. In total, 40 cycles were performed. The cDNA was amplified in duplicate using a Rotor-Gene SYBR Green RT-PCR Kit (Qiagen, Japan) with a Corbett Rotor-Gene RG-3000A Real-Time PCR System. The data were evaluated using the RG-3000A software program (version Rotor-Gene 6.1.93, Corbett). The sequences of the primer pairs are as follows: IL-1β: 5’-CAACCAACAAGTGAT ATTCTCCATG-3’ and 5’-GATCCA CACTCTCCAGCTGCA-3’; TGF-β, 5′-TCAGACATTCGGGAAGCAGTG-3′ and 5′-ATTCCGTCTCCTTGGTTCAGC-3′. For data normalization, an endogenous control (actin) was assessed to control for the cDNA input, and the relative units were calculated with the comparative Ct method.