Isolation and qRT-PCR Analysis of P. aeruginosa Gene Expression

Total RNA was extracted from lung tissue homogenate using TRIzol reagent (Songon Biotech, Shanghai, China) according to the manufacturer’s instructions. Then, cDNA synthesis was conducted using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, K1622, Waltham, MA, United States) in a 20 μl volume of reaction mixture. Real-time PCR was performed in a total reaction volume of 10 μl, containing 5 μl PerfectStartTM Green qPCR SuperMix (TransGen Biotech, Beijing, China), 2 μl template cDNA, 1 μl of primers (Shenzhen Huada Gene Research Institute, Shenzhen, China), and 2 μl DNase/RNase-free water (Tiangen Biotech, Beijing, China). Finally, pvdQ of P. aeruginosa was chosen as the reference gene. The qRT-PCR reaction was conducted on a LightCycler^® 480II Master Mix (Roche, Germany). Data from qRT-PCR experiments were analyzed using the 2^®-ΔΔCT method (Yang et al., 2021 (link)). Primer sequences are listed in Table 1.