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Open Access Highly Accessed Research article

Characterization of human plasma-derived exosomal RNAs by deep sequencing

Xiaoyi Huang1, Tiezheng Yuan1, Michael Tschannen2, Zhifu Sun3, Howard Jacob2, Meijun Du1, Meihua Liang4, Rachel L Dittmar1, Yong Liu5, Mingyu Liang5, Manish Kohli6, Stephen N Thibodeau7, Lisa Boardman6 and Liang Wang1*

Author Affiliations

1 Department of Pathology and Cancer Center, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

2 Human Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

3 Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, 55905, USA

4 Department of Endocrinology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, China

5 Department of Physiology, Medical College of Wisconsi, Milwaukee, WI, 53226, USA

6 Department of Oncology, Mayo Clinic, Rochester, MN, 55905, USA

7 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA

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BMC Genomics 2013, 14:319  doi:10.1186/1471-2164-14-319

Published: 10 May 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.

Keywords:
Exosome; microRNA; Next generation sequencing; Plasma; Biomarker