This study characterized the exosomal RNA profiles in human plasma using deep sequencing. The researchers isolated exosomes from three plasma samples and evaluated the efficacy of three different small RNA library preparation protocols from three manufacturers. They obtained a total of 101.8 million raw single-end reads, with an average of about 7.27 million reads per library. The analysis revealed a diverse collection of exosomal RNA species, with microRNAs (miRNAs) being the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable, and the five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that these highly abundant miRNAs may play important roles in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. The study also detected significant fractions of other RNA species, including ribosomal RNA, long non-coding RNA, piwi-interacting RNA, transfer RNA, small nuclear RNA, and small nucleolar RNA. Additionally, fragments of coding sequence, 5' untranslated region, and 3' untranslated region were also present. The researchers found that the three tested commercial kits generated sufficient DNA fragments for sequencing but had significant biases toward capturing specific RNAs. This study provides a comprehensive characterization of exosomal RNA profiles and contributes to the understanding of exosome-mediated biological functions and mechanisms.This study characterized the exosomal RNA profiles in human plasma using deep sequencing. The researchers isolated exosomes from three plasma samples and evaluated the efficacy of three different small RNA library preparation protocols from three manufacturers. They obtained a total of 101.8 million raw single-end reads, with an average of about 7.27 million reads per library. The analysis revealed a diverse collection of exosomal RNA species, with microRNAs (miRNAs) being the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable, and the five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that these highly abundant miRNAs may play important roles in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. The study also detected significant fractions of other RNA species, including ribosomal RNA, long non-coding RNA, piwi-interacting RNA, transfer RNA, small nuclear RNA, and small nucleolar RNA. Additionally, fragments of coding sequence, 5' untranslated region, and 3' untranslated region were also present. The researchers found that the three tested commercial kits generated sufficient DNA fragments for sequencing but had significant biases toward capturing specific RNAs. This study provides a comprehensive characterization of exosomal RNA profiles and contributes to the understanding of exosome-mediated biological functions and mechanisms.