RT-qPCR analysis of genes encoding components of RND efflux pumps.

The response of P. aeruginosa efflux pump genes to p-anisaldehyde, EGCG, and a combination of both compounds was validated by reverse transcription-quantitative PCR (RT-qPCR). Briefly, 1 μg of total RNA was converted to cDNA using the iScript reverse transcription supermix (Bio-Rad, Hercules, CA, USA) and used in the RT-qPCR assay, performed with the Luminaris Probe qPCR master mix (Thermo Scientific) and oligonucleotide primers and probes targeting mexA, mexC, mexE, mexX, mexK, muxB, and PA1541 (61) (Table 3). Samples of RNA untreated with reverse transcriptase served as a negative control to confirm the absence of contaminating genomic DNA. The analysis was performed with a CFX96 real-time PCR detection system and CFX Maestro software (Bio-Rad). The expression of selected efflux pump genes was normalized to that of the housekeeping gene rpoD.