The standardized method of serum miRNA analysis has been published previously [11 (link)]. Briefly, total RNA was extracted from 300 µL of serum using 3D-Gene® RNA extraction reagent (Toray Industries, Inc., Tokyo, Japan). A comprehensive miRNA microarray analysis was performed using a 3D-Gene® Human miRNA Oligo Chip version 21 (Toray Industries, Inc.). The miRNA signal values were normalized to the ratio of the average signal value of three internal control miRNAs (miR-149-3p, miR-2861, and miR-4463) [12 (link)]. The validity of these control miRNAs was confirmed in the present dataset. To identify robust miRNAs, those with a normalized signal value exceeding 64 in more than 50% of the samples in each group were selected. Data sets analyzed in the study were submitted to the National Center for Biotechnology Information Gene Expression Omnibus database under accession number GSE164174.
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