Differential miRNA Expression Analysis

Continuous and categorical variables were compared using Mann-Whitney U and Fisher’s exact tests, respectively. Unsupervised clustering of normalized miRNA levels was performed using classical multidimensional scale (cmdscale) function. Differential analysis of miRNA expression levels was performed using the limma package [29 (link)]. MiRNA levels with fold changes of ≥ 1 (or ≤ -1) and p-values adjusted for false discovery rate (FDR) of ≤ 0.05 were considered significant. The log2(fold change)|>1|is a widely used threshold used to restrict the analysis to miRNA that are at least two fold up or down regulated to identify meaningful and functionally relevant differences in miRNA expressions. FDR-adjusted p-values were calculated using the Benjamini and Hochberg procedure for multiple testing corrections. Heatmaps were generated using the ‘heatmap’ function, where samples and genes were clustered by the hclust function that uses Euclidian distance. Statistical analysis and data visualization were performed using R software V2.15.0 (http://www.R-project.org) and Bioconductor packages.