fig6

Figure 6. Differential expression analysis of salivary miRNAs across EV isolation methods. (A) Volcano plots showing differentially expressed miRNAs between WS and EV isolates (UC, Q, and M). The x-axis represents log₂FC, and the y-axis indicates statistical significance as -log₁₀(P-value). Red dots mark significantly regulated miRNAs (adjusted P-value < 0.05; |log₂FC| > 1). Right shift (positive log₂FC) indicates upregulation in WS; left shift (negative log₂FC) indicates enrichment in EVs. Grey dots represent non-significant changes. Analyses were performed using DESeq2 (v1.42.1) and visualized with ggplot2 (v3.5.1); (B) Principal component analysis of global miRNA profiles from WS and EV isolates (UC, Q, M), each with three biological replicates. PC1 and PC2 account for 43% and 16% of the total variance, respectively. Analysis was performed using DESeq2; (C) Venn diagram of differentially expressed miRNAs (adjusted P < 0.05) in WS vs. UC (green), WS vs. Q (purple), and WS vs. M (red). Overlaps represent commonly regulated miRNAs across methods. The diagram was generated using the R package VennDiagram (v1.7.3); (D) Heatmap of log₂FCs for 28 miRNAs significantly and commonly regulated across all three comparisons (WS vs. UC, Q, M). Rows represent miRNAs, and columns represent comparisons. The color gradient ranges from red (upregulated in WS) to blue (upregulated in EVs). The heatmap was created using pheatmap (v1.0.12), with log₂FC values scaled from -2 to +2; (E-G) Bar plots showing log₂FCs of all significantly regulated miRNAs in: (E) WS vs. M (n = 47 miRNAs), (F) WS vs. Q (n = 46 miRNAs), (G) WS vs. UC (n = 128 miRNAs). Red bars indicate miRNAs enriched in WS; blue bars indicate those enriched in EVs. Bar plots were created using ggplot2 (v3.5.1); (H-K) Ct values of miRNAs in WS compared to M samples measured by RT-qPCR. miRNAs including hsa-423-5p (H), hsa-193-3p (I), hsa-let7a (J), and hsa-26b (K) are represented with mean ± SEM (n = 9). Statistical analysis was conducted using paired t-tests. ^*P < 0.05 ; ^****P < 0.0001; (L) Comparison of Log₂FCs calculated from NGS and RT-qPCR datasets. WS: Whole saliva supernatant; EV: extracellular vesicles; UC: ultracentrifugation; Q: PEG-based co-precipitation; M: immunoaffinity capture; miRNA: microRNA; log₂FC: log₂ fold change; PCA: principal component analysis; PC1: principal component 1; PC2: principal component 2; DESeq2: differential expression analysis for RNA-seq data; Ct: cycle threshold; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SEM: standard error of the mean; NGS: next-generation sequencing; hsa-miR: Homo sapiens microRNA.