The cellular levels of the corresponding mRNAs were evaluated by real-time PCR. Total RNA was isolated from wild-type or mutant V. vulnificus strains using the RNeasy® Mini Kit and treated with the RNase-free DNase I (TaKaRa). cDNA was synthesized from 4 μg of RNA using the ImProm-IITM RT system (Promega) following the manufacturer’s directions. cDNA was then analyzed with the Light Cycler 480 II Real-Time PCR System (Roche Applied Science) using LightCycler 490 DNA SYBR Green I Master (Roche Applied Science). Real-time PCR was carried out in triplicate in a 96-well plate using the specific primers listed in Additional file 4 : Table S2. The gap gene encoding NAD-dependent glyceraldehyde-3-phosphatase of V. vulnificus was used as an endogenous control for the reactions.
Data are presented as mean ± standard deviation from three independent experiments. Statistical analyses for pair-wise comparison were performed using Student t-test (SYSTAT, SigmaPlot version 11; Systat Software Inc.) to evaluate the statistical significance of the results. Differences were considered significant when P <0.05. Data with P <0.01 are indicated with two asterisks, whereas data with P-values between 0.01 and 0.05 are indicated with a single asterisk.
Data are presented as mean ± standard deviation from three independent experiments. Statistical analyses for pair-wise comparison were performed using Student t-test (SYSTAT, SigmaPlot version 11; Systat Software Inc.) to evaluate the statistical significance of the results. Differences were considered significant when P <0.05. Data with P <0.01 are indicated with two asterisks, whereas data with P-values between 0.01 and 0.05 are indicated with a single asterisk.