Vimentin-p21 mRNA correlation in NB cell lines and tumors - Association between p21 expression and clinical evolution. A. mRNA quantification of vimentin (left panel) and p21 (right panel) relative expression level after GAPDH normalization, in 10 high risk NB cell lines, using Sybr Green Q-PCR. The S- or N-subtype of the NB cell lines is indicated. This result was confirmed after EF1α normalization and by TaqMan Q-PCR quantification, using two different genes for normalization (18 S and TFRC) (data not shown). Bars and error bars represent the means and standard deviations, respectively. B. Representation of the p21 mRNA relative expression level according to that of vimentin, quantified by Sybr Green Q-PCR after 18 S normalization, in 77 NB tumor samples collected at the time of diagnosis (primary tumor biopsies or metastatic bone marrows, all containing more than 70% of tumor cells) (Clinical data are presented in Additional file 1, Table S1). A cDNA sample from the SK-N-SH cell line was used as a reference to integrate all the data together in a unique analysis. The Spearman statistical test was performed with the Graph Pad Prism Software version 4.0. CI: Confidence Interval. C. Left panel: Representation of the p21 mRNA relative expression level according to that of vimentin, quantified by TaqMan Q-PCR after 18 S normalization, in the 40 high risk NB tumor samples of the cohort. The Spearman statistical test was performed as in 1B. This result was obtained using either Sybr-Green or TaqMan technology with two normalization genes, 18 S and GAPDH, or 18 S and TFRC, respectively (data not shown). Right panel: The survival analysis of patients older than one year of age with a stage 4 NB (N = 40) was performed using the Log-rank Kaplan-Meier estimation with the Graph Pad Prism Software version 4.0. The overall survival was defined as the time in months between diagnosis and death or last follow-up (excluding patients who died from toxicity). Patients were separated into two distinct groups according to their p21 mRNA tumor relative expression level quantified by Q-PCR as in the left panel. The median of p21 tumor expression was chosen as a cut-off between high (> median, black line) and low (< median, grey line) expression. HR: Hazard Ratio, CI: Confidence Interval.