Statistical analysis was also made using IBM®SPSS®Statistics software for Windows (Version 22.0). The 2-ΔΔCq method, along with the Student t’ test were used in order to evaluate any statistical differences in the normalized expression levels of the miRNAs here explored. We used Cutoff Finder web application to generate optimum cut-off point for the three deregulated miRNAs in the plasma samples of RCC patients. Cutoff Finder selects the optimum cut-off point, separating patients into high-risk and low-risk groups, by fitting Cox proportional hazard models to the dichotomized variable and the survival variable. The optimal cut-off is defined as the point with the most significant (log-rank test) slip [35 (link)]. A Cox porportional hazard model was used to analyze the patients’ cancer-specific survival, considering as covariants TNM stage, Fuhrman nuclear grade and age (> 60 years). The concordance (χ) was used to compare the predictive ability of the association of well-known prognostic variables with the combined plasma levels of miR-210, miR-221 and miR-1233 with χ > 0.5 being considered with a good prediction ability [27 (link)].
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