Quantitative RT-PCR of E. coli Transcripts

RT-qPCR was performed according to standard procedure [48 (link)]. E. coli cells were inoculated into an LB medium at 37 °C under aeration, with constant shaking at 150 rpm. After inoculation for 24 h, the total RNA was extracted using an ISOGEN solution (Nippon Gene). Total RNAs was transcribed to cDNA with random primers using the THUNDERBIRD SYBR qPCR RT Set (TOYOBO, Osaka, Japan). Quantitative PCR (qPCR) was conducted using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) and a LightCycler 96 system (Roche, Barsel, Switzerland). The primer pairs used are described in Table S1c. The cDNA templates were serially diluted 4-fold and used in the qPCR assays. The qPCR mixtures, each 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 cycle conditions: 95 °C treatment for 2 min, at 45 cycles of 10 s at 95 °C, and 20 s at 55 °C, and then incubation for 20 s at 72 °C. The 16S rRNA expression level was used to normalize the varying levels of the test samples, and the relative expression levels were quantified using the Relative Quantification software provided by Roche. The results are presented as the average of three independent experiments.