2.7. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR)
After various treatments, the total RNA of each group of cells was extracted with Trizol reagent and reverse-transcribed according to the instructions of the reverse transcription PCR kit (Servicebio, China). Then, amplification was performed using qPCR SYBR Green Master Mix Reagent (Yeasen Biotechnology, China). The relative expression levels of mRNA in each sample were assessed using the 2−ΔΔCtmethod and normalized to GAPDH expression. Primer design and synthesis are as follows:
NLRP3 forward primer: 5’-GAGGAAAAGGAAGGCCGACA-3’.
NLRP3 reverse primer: 5’-TGGCTGTTCACCAATCCATGA-3’.
Caspase-1 forward primer:5’-AGACATCCCACAATGGGCTC-3’.
Caspase-1 reverse primer: 5’-TGAAAATCGAACCTTGCGGAAA-3’.
IL-18 forward primer: 5’-ACTGTAGAGATAATGCACCCCG-3’.
IL-18 reverse primer: 5’-AGTTACAGCCATACCTCTAGGC-3’.
IL-1β forward primer:5’-GAGCAACAAGTGGTGTTCTCC-3’.
IL-1β reverse primer: 5’- AACACGCAGGACAGGTACAG-3’.
GAPDH forward primer:5’-CGTGGAAGGACTCATGACCA-3’.
GAPDH reverse primer: 5’-GGCAGGGATGATGTTCTGGA-3’.