RT-qPCR Analysis of E. coli Gene Expression

RT-qPCR analysis was performed according to standard procedures [43 (link)]. E. coli cells were inoculated into M9 minimal medium supplemented with CAA (0.2 %) at 37 °C with aeration by constant shaking at 150 r.p.m. Total RNA was extracted from exponential phase E. coli cells (OD₆₀₀=0.4) using ISOGEN solution (Nippon gene, Tokyo, Japan). Total RNA was transcribed to cDNA with random primers using the THUNDERBIRD SYBR qPCR RT Set (TOYOBO, Osaka, Japan). Quantitative PCR (qPCR) was conducted using THUNDERBIRD SYBR qPCR Mix (TOYOBO) and a LightCycler 96 system (Roche, Basel, Switzerland). The primer pairs used are described in Table S1b. The cDNA templates were serially diluted four-fold and used in qPCR. The qPCR mixtures, containing 10 µl of THUNDERBIRD SYBR qPCR Mix (TOYOBO), 1 µl of each primer (5 µM stock), 7 µl of water, and 1 µl of cDNA, were amplified under the following thermal cycling conditions: 2 min at 95 °C, 45 cycles of 10 s at 95 °C and 20 s at 55 °C, and then incubated for 20 s at 72 °C. The 16S rRNA expression level was used to normalise the varying levels of the test samples, and the relative expression levels were quantified using Relative Quantification software provided by Roche. The results are presented as the average of three independent experiments.