title: Characterization of human plasma-derived exosomal RNAs by deep sequencing.
abstract: BACKGROUND
  Exosomes, endosome-derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell-cell communication. These RNA transcripts have great potential as disease biomarkers. To characterize exosomal RNA profiles systemically, we performed RNA sequencing analysis using three human plasma samples and evaluated the efficacies of small RNA library preparation protocols from three manufacturers. In all we evaluated 14 libraries (7 replicates).
  
  
  RESULTS
  From the 14 size-selected sequencing libraries, we obtained a total of 101.8 million raw single-end reads, an average of about 7.27 million reads per library. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which microRNAs (miRNAs) were 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. The five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) collectively accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that the highly abundant miRNAs may play an important role in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. From the unknown RNA sequences, we predicted 185 potential miRNA candidates. Furthermore, we detected significant fractions of other RNA species including ribosomal RNA (9.16% of all mappable counts), long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%); fragments of coding sequence (1.36%), 5' untranslated region (0.21%), and 3' untranslated region (0.54%) were also present. In addition to the RNA composition of the libraries, we found that the three tested commercial kits generated a sufficient number of DNA fragments for sequencing but each had significant bias toward capturing specific RNAs.
  
  
  CONCLUSIONS
  This study demonstrated that a wide variety of RNA species are embedded in the circulating vesicles. To our knowledge, this is the first report that applied deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.
authors.affiliation: Department of Pathology and Cancer Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Xiaoyi
authors.initials: X
authors.lastname: Huang
authors.forename: Tiezheng
authors.initials: T
authors.lastname: Yuan
authors.forename: Michael
authors.initials: M
authors.lastname: Tschannen
authors.forename: Zhifu
authors.initials: Z
authors.lastname: Sun
authors.forename: Howard
authors.initials: H
authors.lastname: Jacob
authors.forename: Meijun
authors.initials: M
authors.lastname: Du
authors.forename: Meihua
authors.initials: M
authors.lastname: Liang
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Dittmar
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Mingyu
authors.initials: M
authors.lastname: Liang
authors.forename: Manish
authors.initials: M
authors.lastname: Kohli
authors.forename: Stephen N
authors.initials: SN
authors.lastname: Thibodeau
authors.forename: Lisa
authors.initials: L
authors.lastname: Boardman
authors.forename: Liang
authors.initials: L
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D001782:Blood Donors; D002874:Chromosome Mapping; D055354:Exosomes / Q000235:genetics*; D005110:Extracellular Space / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics; D010949:Plasma / Q000166:cytology*; D020871:RNA Stability; D017423:Sequence Analysis, RNA; D059467:Transcriptome
chemical_list: D035683:MicroRNAs