Characterization of human plasma-derived exosomal RNAs by deep sequencing - PubMed (original) (raw)

doi: 10.1186/1471-2164-14-319.

Tiezheng Yuan, Michael Tschannen, Zhifu Sun, Howard Jacob, Meijun Du, Meihua Liang, Rachel L Dittmar, Yong Liu, Mingyu Liang, Manish Kohli, Stephen N Thibodeau, Lisa Boardman, Liang Wang

Affiliations

Characterization of human plasma-derived exosomal RNAs by deep sequencing

Xiaoyi Huang et al. BMC Genomics. 2013.

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.

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Figures

Figure 1

Figure 1

Exosome isolation and exosomal small RNA quantification. (A) Representative histogram of exosome size distribution. (B) Exosomal RNAs determined using the Agilent Small RNA Chip. The small RNAs were dominant in the exosomal RNAs. (C) Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples A, B and C contained no detectable 18S and 28S rRNAs. Cellular RNA from HEK293 was loaded as positive control for 18S and 28S rRNAs (D) Isolated exosomal RNAs treated either with DNase I or RNase A. (E) Plasma and control small RNAs treated under various conditions.

Figure 2

Figure 2

Library preparation and analysis of the raw sequencing data. (A) PAGE analysis of the sequencing libraries prepared using three different kits. 20-bp DNA ladders are shown on the left of each panel while the prepared libraries are shown on the right. The anticipated RNA sequencing constructs ranging from 140–160 bp are highlighted within brackets. The DNA bands (~125 bp) are the adaptor dimers. (B) Percentage of mappable RNAs in the raw sequencing reads. (C) Abundance of the selected miRNAs and β-actin in plasma exosomal RNA and HEK293 cellular RNA. Gene and miRNA abundance was determined by the threshold cycle (Ct), where Ct values >30 are defined as rare. (D) Percentage of each miRNA in the repertoire of total miRNA reads.

Figure 3

Figure 3

miRNA correlations between technical replication, biological replication and methodological replication. (A) Scatter plots of technical replicates in sample A. Illumina kit (ILMN), left panel (r = 0.978); Bioo Scientific kit (BS), middle panel (r = 0.984); and NEB kit, right panel (r = 0.994). (B) Scatter plots of sample A and sample B (left panel, r = 0.983); sample B and C (middle panel, r = 0.986), and sample A and C (right panel, r = 0.981). Only the plots for the pooled samples are shown. (C) Scatter plots of ILMN and BS (left panel, r = 0.889), ILMN and NEB (middle panel, r = 0.866), and BS and NEB (right panel, r = 0.898). Only the plots for the pooled samples are shown. (D) Heat map of unsupervised hierarchical clustering of the 100 most abundant miRNAs. Samples and library preparation methods are indicated on horizontal axis at the top of the heat map. miRNAs are indicated vertically on the right.

Figure 4

Figure 4

Venn diagrams showing miRNAs that are common in the three samples. (A) Unique and shared miRNAs in sample A in the libraries prepared using the different kits. (B) Unique and shared miRNAs in the different samples. The miRNAs with read counts ≥5 per million mappable sequences were used for the comparison.

Figure 5

Figure 5

Other RNA species that were detected in the exosomal RNA libraries. (A) Pie chart of RNA species and their distributions in the plasma-derived exosomes. Misc RNAs are the RNA sequences that mapped to the human genome but not in any of the categories listed. The DNA category represents the novel transcripts that have no annotation in the human RNA database. (B) Graphic and statistics of a representative novel miRNA predicted by miRDeep2. Both star and mature strands were detected and integrated. Lower left table shows information about the sample and the miRDeep2 scores, along with the read count for each component of the putative miRNA. mm, number of mismatches. Mismatched nucleotides are indicated by uppercase letters.

Figure 6

Figure 6

Schematic illustration of the study design. Three plasma samples (A, B and C) were used in this study. Each sample had a technical replicate. Three commercial kits were tested: ILMN-Illumina, BS-Bioo Scientific and NEB-New England Biolab.

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