Small non-coding RNAs (ncRNAs) function as bioactive molecules packaged inside extracellular vesicles (EVs), where they influence a broad spectrum of physiological and disease-related pathways. This investigation presents an in-depth expression atlas of seven distinct categories of small ncRNAs in EVs isolated from both serum and plasma across multiple conditions. Large EVs (lEVs) and small EVs (sEVs) alike contain considerable fractions of miRNAs (∼28.2% in lEVs and ∼20.8% in sEVs) and ribosomal RNAs (∼24.0% in lEVs and ∼19.1% in sEVs). Compared with sEVs, lEVs show markedly higher transfer RNA content (∼38.8%), whereas sEVs are characterized by elevated Y RNA levels (∼22.5%). In particular, Y RNA abundance rises substantially in sEVs obtained from older donors (age ≥60 years), an observation not replicated in lEVs. Serum-derived EVs display greater variety in their small ncRNA repertoire than plasma-derived counterparts. A substantial overlap (>50%) is evident among the 100 most abundant small ncRNAs shared between lEVs and sEVs. The miRNAs hsa-miR-16-5p, hsa-let-7a-5p, hsa-miR-142-3p, and hsa-miR-103-3p rank consistently within the ten most highly expressed small ncRNAs in both plasma- and serum-derived lEVs, as well as in peripheral blood mononuclear cells. In addition, serum-derived sEVs from patients with glioblastoma, breast cancer, prostate cancer, and gastric cancer are enriched in distinctive, highly abundant miRNAs, snoRNAs, small nuclear RNAs, and piRNAs. Collectively, these observations delineate the distinctive cargo signatures of small ncRNAs within lEVs and sEVs from serum and plasma under normal and pathological states, thereby supplying useful reference points for subsequent biomarker development and therapeutic exploration.
