2.9 Quantitative PCR (qPCR)
Total RNA was extracted from the top and bottom layers of the chip using an RNA extraction kit (cat no. AG21023, Accurate bio, China) and subsequently converted to cDNA using a reverse transcription kit (cat no. AG11707, Accurate bio, China), with inputs ranging from 50 to 500 ng of RNA. The expression levels of inflammatory cytokines and other genes were assessed by fluorescence-based qPCR. Primers information was provided in Table 1. PCR amplification involved an initial step of 50°C for 10 minutes, denaturation at 95°C for 1 minute, followed by 45 cycles of 95°C for 15 seconds, and 60°C for 45 seconds. Expression data were quantified employing the 2-∆∆Ct method.