Mx3000p pcr detection system

To validate of the reliability of transcriptome sequencing data, real-time quantification of random mRNAs was performed using qRT-PCR with the same total cellular RNAs used for transcriptome experiments. We randomly selected 28 genes for qRT-PCR validation, i.e., 16 representative downregulated genes and 12 representative upregulated genes in the ΔtrxA mutant. Specific primers for these genes were designed and purchased from Sangon Biotech. cDNA was synthesized with reverse transcriptase (Toyobo). Quantitative PCR was performed in 20 μL reaction mixtures containing SYBR quantitative PCR mix (Toyobo) to measure the transcriptional levels of genes of interest using the Mx3000P PCR detection system (Agilent) with specific primer pairs. Real-time quantitative PCR was performed in a 20 μL reaction volume containing 200 ng cDNA, 10 μL SYBR quantitative PCR mix and 1 μL gene-specific primers (200 nM). The housekeeping gene, rpoB, was used as an internal control for normalization in each sample. Triplicate assays were performed for each gene.

Listeria monocytogenes wild-type 10403S and its mutant strain ΔArgR were grown to the stationary phase (OD_600nm = 1.2) in BHI broth at 37°C, and then exposed to acidic (pH 5.5) and neutral (pH 7.0) conditions, respectively, for additional 1 h. Total RNA was extracted using the Column Bacterial total RNA Purification Kit (Sangon), according to the manufacturer’s instructions, genomic DNA removed using DNase I (TaKara, Japan) and cDNA synthesized with reverse transcriptase (TOYOBO, Osaka, Japan). Real-time quantitative PCR was performed in a 20 μL reaction volume containing 200 ng cDNA, 10 μL SYBR quantitative PCR mix (TOYOBO), and 1 μL gene-specific primers (200 nM, Supplementary Table S1) to measure the transcriptional levels of arcA, sigB, argC, and argG using the Mx3000P PCR detection system (Agilent). The housekeeping gene, gyrB, was used as an internal control for normalization in each sample as previously described (Chen et al., 2011 (link)). Relative transcription levels were quantified using the 2^-ΔΔCT method and shown as relative fold changes (Livak and Schmittgen, 2001 (link)). Triplicate assays were performed for each gene.

To further establish, whether Grx is involved in bacterial virulence, qRT-PCR was employed to analyze the changes in transcripts of the major virulence-associated genes, prfA, hly, plcA, plcB, mpl, inlA and inlB. Bacteria grown to the stationary phase were exposed to the oxidative stress (4 mM diamide) for 1 h. Total RNA was extracted using the Column Bacterial total RNA Purification Kit (Sangon), according to the manufacturer’s instructions, genomic DNA removed using DNase I (TaKaRa) and cDNA synthesized with reverse transcriptase (TOYOBO). Real-time quantitative PCR was performed in a 20 µL reaction volume containing 200 ng cDNA, 10 µL SYBR quantitative PCR mix (TOYOBO), and 0.5 µL gene specific primers (200 nM, Supplementary Table S1) to measure the transcriptional levels of specific genes using the Mx3000P PCR detection system (Agilent). The housekeeping gene, 16SrRNA, was used as an internal control for normalization. Relative transcription levels were quantified using the 2^−ΔΔCT method and shown as relative fold changes. Triplicate assays were performed for each gene.