*4.6. Bioinformatics Analysis*

After data processing, expression statistics for the normalized reads were evaluated using analysis of variance to identify differentially expressed genes. Differentially expressed genes were selected if the fold changes (FC) in expression was > 1.5 with a *p*-value < 0.5. From the data set of miRNAs and mRNAs for which expression was significantly altered between serum exosomes isolated from young and old rats, the potential regulation of a mRNA by a particular miRNA was predicted with TargetScan (http://www.targetscan.org/, MIT, MA, USA). The paired miRNAs and mRNAs were used for further analysis.

Hierarchical cluster analysis (HCA) is an algorithmic approach to identify groups with varying degrees of (dis)similarity in a data set represented by a (dis)similarity matrix. This analysis was carried out with the Pheatmap package (https://CRAN.R-project.org/package=pheatmap, Estonia). The volcano plot is a type of scatter-plot that can quickly identify changes in individual data in large data-sets composed of replicate data; ggplot2 package (http://ggplot2.org, New Zealand) was used for this purpose.
