Relative expression software tool rest 2009 v2

The amounts of dltA transcripts in Bt407 and ΔdltX strains were measured by real time reverse transcription. RNA extraction and cDNA synthesis were performed as described previously (Réjasse et al., 2012 (link)). Primers BCF1372.Q3 and BCR1372.Q3 located inside the dltA gene were designed with Primer Express software from Applied Biosystems. Real time PCR was carried out with Sybr green PCR master mix (Applied Biosystems) as recommended by the supplier. Mean values were calculated from two separate experiments in which qPCR reactions were performed in triplicate. The cycle threshold was used to determine the relative dltA gene expression levels in the two genetic backgrounds. Data were analyzed by the comparative threshold cycle (ΔΔCt) method with the Relative Expression Software Tool (REST 2009 V2.0.13, QIAgen). Expression ratios were normalized to two Bt 407 endogenous reference housekeeping genes, pur and tpi.

Results were expressed as mean + SD from four independent experiments. Influence of CCN1-Fc on cell viability was calculated by nonparametric Kruskal-Wallis analysis with Dunn’s post-hoc multiple comparison test using GraphPad Prism 6.03 (GraphPad Software, CA, USA). P values less than 0.05 were considered to be statistically significant. Significances of qPCR analysis was calculated with the Relative Expression Software Tool (REST 2009 V2.0.13) obtained from Qiagen GmbH (Hilden, Germany)
[39 (link)].

For all quantitative datasets, the bars represent the mean with error bars representing the SEM from a total of n replicates in N independent experiments. After considering normality, datasets involving a control group and a treatment group were analyzed on GraphPad Prism 7 (RRID: SRC_002798) by using unpaired, two-tailed Student’s t test. Datasets involving a control group and multiple treatment groups were analyzed on GraphPad Prism 7 using one-way ANOVA with Dunnett’s or Bonferroni’s post hoc test. RT-qPCR data were analyzed on the Relative Expression Software Tool (REST) 2009 V2.0.13 (QIAGEN). The expression of spry1, sema3a, and slit1/2 were normalized against the stably expressed reference genes actb, tubb, dynll1. For all statistical analyses, the threshold of significance was set at 0.05. The details of the statistical analyses are shown in Table 2.