Figure 1.

Technical pipeline for identification of plasma exosomal miRNAs by next-generation sequencing. (A) Representative size distribution of exosomes isolated from plasma determined using nano-flow cytometry. (B) Representative particle concentration of exosomes isolated from plasma determined by nano-flow cytometry. (C) Western blot showing the expression level of the typical exosome markers Calnexin (negative in exosomes), TSG-101, CD63, and CD81(positive in exosomes) in the four randomly selected exosomal samples from each group. S1–4, sample 1–4. PC, positive control using whole cell lysate prepared from HEK 293 cells. (D) Transmission electron microscopy picture showing the morphology of the exosomes. Scale bar on the left represents 200 nm, on the right 100 nm. (E) Polyacrylamide gel electrophoresis (PAGE) analysis for the established small RNA libraries. DNA ladders are shown in the 1st, 4th, and 7th lanes of the panel, while the DNA bands of prepared libraries are shown between the marker lanes. The anticipated size of the RNA sequencing constructs is ~150 bp. The DNA bands corresponding to 130 bp are the adaptor dimmers. M, DNA ladder. L1–4, library 1–4. (F) High-sensitivity DNA chip run on Agilent 2100 bioanalyzer demonstrates the quality and quantity of the gel-recovered library. (G) Illustrated RNA species and their abundance in the raw reads of exosomal RNA. Mappable reads are the sequences that are mapped to known human RNAs. lncRNA, long noncoding RNA; miRNA, microRNA; piRNA, piwi-interacting RNA; rRNA, ribosomal RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; tRNA, transfer RNA. (H) Top 30 known miRNAs with the highest expression levels in exosomal miRNA libraries. (I) Top 10 novel miRNAs predicted by miRDeep2 and miREvo with the highest expression levels in exosomal miRNA libraries.