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’.