Control mirna assay

The RNA extracted with the mirVana kit was used. cDNA was synthesized using the TaqMan microRNA RT kit (Applied Biosystems) following the manufacturer’s instructions. A total of 10 ng of RNA was used in the reverse transcription reaction with miRNA-specific primers. The real-time reactions were performed in a 7500 high-throughput (HT) fast real-time PCR system (Applied Biosystems). Data were analyzed using the comparative threshold cycle (2^−ΔΔCT) method. The threshold cycle (C_T) values for the selected miRNA targets were subtracted from the C_T values of the endogenous small noncoding RNA control RNU6B (control miRNA assay; Applied Biosystems).

The analysis procedures were performed in the R environment (version 3.1.1) 31 . The normalization step was performed according to the 2^-ΔΔCt method 32 (link). Cycle threshold (Ct) values from the selected miRNA targets were subtracted from the Ct values of endogenous small non-coding RNA control RNU6 (Figure 1) (Control miRNA Assay, Applied Biosystems). A subsequent ΔΔCt value was calculated for the malignant tumors using the lower expression values of the control or benign ΔCt values. Expression values are represented as the ΔΔCt value on a log2 scale. Statistical analysis was performed using one-way ANOVA with a significance level of p≤0.05. To assess the sensitivity and specificity of each biomarker, ROC curve analysis was used. Analysis of the combination of biomarkers was performed using a general logistic model (glm). ROC analysis was performed using the ROCR package 33 .