Improm iitm rt system

The mRNA levels were determined by qRT-PCR following previous study^{50 (link)}. Total RNA from cells or from xenografted tumor was isolated by TRIzol® (15596-026, Invitrogen, Carlsbad, CA, USA). The RNA is used to confirm TRAF4, NOX2, NOX4 and ICAM1 mRNA levels after each treatment. To obtain cDNA from isolated mRNA, the isolated mRNA was utilized by reverse transcription (RT) reaction, which was conducted on an ImProm-IITM RT system (A3800, Promega, Madison, WI, USA) following the manufacturer’s protocol. Primers used for mRNA expression are listed in Supplementary Table 1. RT reaction conditions were 25 °C for 5 min, 42 °C for 60 min, 85 °C for 5 min, and 4 °C for overnight. A SYBR Green core reagent kit (4367659, Applied Biosystems, Foster City, CA, USA) and a real-time PCR plate (N8010560, Applied Biosystems) were used for performing qRT-PCR, which was performed by using the Applied Biosystems-7900 HT qRT-PCR instrument (Applied Biosystems). The qRT-PCR conditions were 40 cycles of 15 s at 95 °C and 1 min at 60 °C, which was followed by thermal denaturation. Each mRNA level was measured in triplicate. In addition, each mRNA level was normalized by the GAPDH mRNA level and calculated by using the 2^−ΔΔCT method. To simplify data presentation, relative expression values were multiplied by 10².

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-II^TM 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.