*4.10. Measurement of mRNA Expression Levels of Inflammatory Mediators*

To investigate the cytokines mRNA expression of L5-treated monocyte/macrophage, we extracted RNA by the TRI Reagent method (Sigma-Aldrich). The High-Capacity cDNA Reverse Transcriptase Kit (ThermoFisher) was used to reverse-transcribe 1 μg RNA into cDNA using for qRT-PCR analyses. The expression levels of each gene were determined by the CFX96 Real-time PCR system (BioRad, Hercules, USA) with IQ SYBR Green Supermix reagent (BioRad). PCR using 10 ng cDNA was performed with one cycle of preincubation at 95 ◦C for 3 min, 45 cycles of amplification (95 ◦C for 15 s, 60 ◦C for 1 min), and the melt curve detection program from 55 ◦C to 95 ◦C. The primer sequences are as follows: IL-6, 5 -AGACAGCCACTCACCTCTTCAG-3 (forward) and 5 -TTCTGCCAGTGCCTCTTTGCTG-3 (reverse); IL-8, 5 -GAGAGTGATTGAGAGTGGACCAC-3 (forward) and 5 -CACAACCCTCTGC ACCCAGTTT-3 (reverse); TNF-α, 5 -CCACTTCGAAACCTGGGATTC-3 (forward) and 5 -TTAGTGGTTGCCAGCACTTCA-3 (reverse). The mRNA expression levels of cytokines were calculated using the comparative threshold cycle (Ct) method and were evaluated by 2−Ct, Ct = CtTarget gene–CtActin. The results were normalized to the levels of actin mRNA and were expressed relative to the levels in control cells (relative value = 1).
