Characterization of human plasma-derived exosomal RNAs by deep sequencing

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

Research output: Contribution to journalArticle

393 Citations (Scopus)

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.

Original languageEnglish (US)
Article number319
JournalBMC Genomics
Volume14
Issue number1
DOIs
StatePublished - May 10 2013

Fingerprint

RNA Sequence Analysis
High-Throughput Nucleotide Sequencing
RNA
MicroRNAs
Libraries
Biomarkers
Small Nucleolar RNA
Long Noncoding RNA
Exosomes
RNA Splicing
Small Nuclear RNA
Ribosomal RNA
5' Untranslated Regions
Endosomes
3' Untranslated Regions
Transfer RNA
DNA Sequence Analysis
Cell Communication
Chromosome Aberrations
Small Interfering RNA

Keywords

  • Biomarker
  • Exosome
  • MicroRNA
  • Next generation sequencing
  • Plasma

ASJC Scopus subject areas

  • Biotechnology
  • Genetics

Cite this

Huang, X., Yuan, T., Tschannen, M., Sun, Z. D., Jacob, H., Du, M., ... Wang, L. (2013). Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics, 14(1), [319]. https://doi.org/10.1186/1471-2164-14-319

Characterization of human plasma-derived exosomal RNAs by deep sequencing. / Huang, Xiaoyi; Yuan, Tiezheng; Tschannen, Michael; Sun, Zhifu D; Jacob, Howard; Du, Meijun; Liang, Meihua; Dittmar, Rachel L.; Liu, Yong; Liang, Mingyu; Kohli, Manish; Thibodeau, Stephen N; Boardman, Lisa Allyn; Wang, Liang.

In: BMC Genomics, Vol. 14, No. 1, 319, 10.05.2013.

Research output: Contribution to journalArticle

Huang, X, Yuan, T, Tschannen, M, Sun, ZD, Jacob, H, Du, M, Liang, M, Dittmar, RL, Liu, Y, Liang, M, Kohli, M, Thibodeau, SN, Boardman, LA & Wang, L 2013, 'Characterization of human plasma-derived exosomal RNAs by deep sequencing', BMC Genomics, vol. 14, no. 1, 319. https://doi.org/10.1186/1471-2164-14-319
Huang, Xiaoyi ; Yuan, Tiezheng ; Tschannen, Michael ; Sun, Zhifu D ; Jacob, Howard ; Du, Meijun ; Liang, Meihua ; Dittmar, Rachel L. ; Liu, Yong ; Liang, Mingyu ; Kohli, Manish ; Thibodeau, Stephen N ; Boardman, Lisa Allyn ; Wang, Liang. / Characterization of human plasma-derived exosomal RNAs by deep sequencing. In: BMC Genomics. 2013 ; Vol. 14, No. 1.
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AU - Jacob, Howard

AU - Du, Meijun

AU - Liang, Meihua

AU - Dittmar, Rachel L.

AU - Liu, Yong

AU - Liang, Mingyu

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N2 - 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.

AB - 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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KW - Exosome

KW - MicroRNA

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