Quantitative PCR and RNA-Seq analysis of serum sample from NPC patients. Quantitative PCR was used to determine relative expression levels for 40 miRNAs in 40 sera samples comprised of 14 control samples and 26 test cases; 16 sera from Malaysian patients, 16 from German patients and eight from American patients (Malaysian, German, and American). A) Volcano plots of qPCR analysis of 40 sera samples. A single miRNA (miR-486-5p) was found to be significantly up-regulated when samples were analyzed as a whole (panel one; All). An analysis of Malaysian sera was also conducted incorporating anti-viral capsid antigen (VCA) IgG titers divided into four groups of four; a control group, low VCA titer group (second panel; Malaysian (40–160)), a medium VCA titer group (third panel; Malaysian (320–640)) and a high VCA group (fourth panel; Malaysian (> 640). Blue lines indicate a p-value of 0.05 and dashed lines show a four fold-change. Dysregulated miRNAs are shown in blue and labeled with the human mature miRNA identification number. B) Heatmap of significantly dysregulated miRNAs from RNA-Seq analysis of Malaysian sera. The heatmap was generated using the ‘heatmap.2′ function in the gplots R package and miRNA counts per million values were scaled across samples and colored to represent up-regulation (1.0) and down-regulations (−1.0). C) Comparison of FC values, generated using RNA-Seq, for miRNAs identified in FFPE and sera when compared to their respective controls. Left panel (All) shows comparison for all miRNAs identified in both analyses and the right panel (Significant in Both) for miRNAs identified as dysregulated in both tissues. Three miRNAs were identified as significantly up-regulated in sera but significantly down-regulated in FFPE tissue. Asterisk significant dysregulation in microarray analysis but not RNA-Seq. D) Comparison of FC values by qPCR and RNA-Seq in sera. Only miRNAs that were significantly dysregulated in RNA-Seq and were present on the custom qPCR chip are shown.