For array data analysis, SDS Software v.2.3 (Thermo Fisher Scientific) was used to calculate Raw Ct values, with an automatic baseline and threshold. ExpressionSuite Software 1.1 (Thermo Fisher Scientific) was used to calculate RQ (2-∆∆Ct) values. Data were normalized using global normalization, an algorithm that finds the assays common to every sample and then uses the median Ct of those assays as the normalization factor, on a per sample basis [25 (link)]. Ct values > 35 or with Amp score < 0.7 were excluded from the analysis. To identify candidate miRNAs differentially expressed between patients and controls, we selected miRNAs with low p values (p ≤ 0.05). P values were calculated by the ExpressionSuite software using Student’s t-test for sample group comparisons, without multiple test correction. Further to this, we screened the group for miRNAs expressed in every sample and with low variability among the same group.
For qRT-PCR data analysis, Excel software (Microsoft Office 365 ProPlus) was used to calculate ∆Ct, −∆∆Ct, and RQ for patients and controls. Statistical analysis was performed on RQ values through the demo version of GraphPad Prism 6.01 software using an unpaired non-parametric two-sided Mann-Whitney test. Confidence level was set at 95% (p value ≤0.05).
For qRT-PCR data analysis, Excel software (Microsoft Office 365 ProPlus) was used to calculate ∆Ct, −∆∆Ct, and RQ for patients and controls. Statistical analysis was performed on RQ values through the demo version of GraphPad Prism 6.01 software using an unpaired non-parametric two-sided Mann-Whitney test. Confidence level was set at 95% (p value ≤0.05).
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