Overview
Extracellular vesicles (EVs) of 40-100 nanometers in diameter are continuously secreted by many cells, and are found in most biofluids such as blood, urine, breast milk, semen, amniotic and ascites fluids. EVs can be categorized as microvesicles or exosomes depending on their origin from either direct shedding of vesicles from the plasma membrane or from multivesicular endosomes, respectively1. Exosomes are lipid-rich small vesicles that have been shown to transport biomolecules such as nucleic acid and proteins between cells and in some cases serve as a platform for intercellular communication and disease transmission. Since exosomes and microvesicles are difficult to distinguish and to separate using lab processes, we use the terms EVs and exosomes herein to represent biofluid-derived vesicles of the above mentioned size range. Through the advancement of screening technologies, exosomes are now considered to be reliable sources for circulating early diagnostic biomarkers2,3. Visit ORB's exosomal RNA overview page for more information on exosome function and role in pathogenesis.

Messenger RNA (mRNA) constitutes one type of genetic information that is carried by exosomes, and the delivery of mRNA via exosomal transmission can result in fully functional or translatable long RNA within a recipient cell4-6.  Exosomal mRNA has been determined to be useful as biomarkers from blood-based, saliva, and urine biofluid samples7.8. Long noncoding RNAs (lncRNA) are also present in exosomes and can show expression patterns that are distinct from the cellular level9,10.

Exosome mRNA Profiling at ORB
ORB exosome mRNA characterization service includes isolation of extracellular vesicles and extraction of their associated RNA using commercial kits based on affinity purification such as Qiagen's ExoRNeasy and Norgen's Plasma/ Serum Exosome Purification and RNA Isolation kit. Isolation is possible from most biofluid samples such as serum, plasma, saliva, urine, semen, breast milk, and cerebrospinal fluid; contact us if you would like to profile a biofluid not included on this list.  ORB provides RNA isolation from purified exosomes and quality assessment of the purified RNA by real-time PCR for housekeeping genes as well as by electrophoresis using an Agilent Bioanalyzer RNA PicoChip. 

ORB provides exosomal long RNA profiling of cell-free biofluid and PAXgene whole blood samples.

Profiling of mRNA from whole blood samples collected in PAXgene tubes is also available; this, however, provides for examination of RNA derived from unfractionated whole blood rather than isolated EVs. The RNA in these samples is primarily derived from leukocytes such as B and T lymphocytes, monocytes, ensinophils, basophils, and neutrophils. PAXgene tubes contain a compound that maintains the integrity of RNA in whole blood during processing and storage, even under non-optimal storage conditions (several days at room temp). Using PaxGene tubes it is possible to obtain very reproducible measurements of gene expression because pre-isolation of specific blood cells is not required prior to RNA extraction.

Although long RNA from whole blood includes some RNA from exosomes, whole blood RNA analysis does not facilitate the focused identification and measurement of the levels of mRNAs that are strictly associated with EVs. Thus, ORB's exosome-focused service is most appropriate for clients wishing to identify molecules that are released into the blood stream within EVs, which is particularly relevant for studies of intercellular communication, disease pathogenesis, and biomarker discovery. Alternatively, ORB also provides RNA sequencing of buffy coat, PBMCs, and flow-sorted lymphocytes, for studies targeting gene expression in specific cells of the immune system.

From purified exosomal RNA, ORB typically prepares ribosomal-depleted strand-specific DNA libraries containing both polyA+ and polyA- sequences using the Clontech SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian.  For more information about additional library preparation options visit ORB's long RNA sequencing page.  Sequencing of DNA libraries prepared from exosomal mRNA samples is accomplished using the Illumina NextSeq 500, HiSeq 2500 or HiSeq 4000 instruments.  

ORB's bioinformatic services supporting exosomal mRNA characterization include differential expression and statistical analyses that are customized to each project's experimental goals. Learn more about ORB's long RNA sequencing bioinformatics services. Additional data analysis services include analysis of long noncoding RNAs which are implicated in many diseases and predictive modeling support to facilitate development of marker sets useful for companion diagnostics, prediction of disease severity, patient stratification and other applications. The exosomal RNA profiling overview page provides examples of successful projects wherein ORB's exosome RNA profiling services were used to advance research projects in specific disease areas. Contact us if you would like to discuss how ORB's exosomal long RNA profiling can advance your research objectives.

Sample Submission Instructions

ORB provides exosomal mRNA examination of cell-free biofluid and PAXgene samples.  Some examples of cell-free biofluid samples ORB routinely handles include:

  • Serum
  • Plasma
  • Cerebrospinal Fluid
  • Bronchial Lavage
  • Saliva
  • Urine
  • Ascites Fluid
  • Semen
  • Milk

Contact us about processing with other sample types.

Sample preparation instructions, minimum volume requirements, and sample submission checklists for each sample type are linked from Table 1, below.

Table I. Biofluid volume requirements and sample submission documents for exosomal mRNA sequencing.
Sample Type Fraction Required Volume (ml) Preparation
Instructions
Sample Submission Checklist
Cell-free biofluid (e.g. plasma) Microvesicle/
exosome
1.0 Biofluid - exosome mRNA
Whole Blood Total 1 PAXgene tube PAXgene tubes

Exosomal mRNA isolation services available using biofluid samples. Contact us about submission of other sample types. ORB is a Biosafety Level 2 (BSL-2) laboratory, and can accept samples that contain agents associated with human diseases.
 

Demonstration of ORB's Long RNA Sequencing Service for Exosomes

Sample Processing

Table II. Comparison of tissue- and serum-exosome RNA using ORB's long RNA sequencing workflow.
Sample Type Input
Serum ExoRNA - Patient #7 1.0 ml equiv.
Serum ExoRNA - Patient #7 1.0 ml equiv.
Serum ExoRNA - Patient #8 1.0 ml equiv.
Serum ExoRNA - Patient #8 1.0 ml equiv.
Tissue Total RNA 250 pg
Tissue Total RNA 1 ng
Tissue Total RNA 10 ng

To compare tissue- and serum-exosome RNA, RNA was isolated from human serum exosomes using the Qiagen ExoRNeasy Kit (see Table II). Stranded, rRNA-depleted cDNA libraries were prepared from the DNA-free total RNA. Approximately 35 million passed-filter single-end reads per sample were generated by long RNA sequencing using the Illumina HiSeq-2500 instrument, and data analysis was performed with ORB long RNA bioinformatics pipeline.


Exosomal mRNA Profiling Results

Fig. 1 Serum exosome biotype composition by sample type.

Serum exosome samples had 60-70% of trimmed reads map to hg38 genome, while tissue samples had 80-89% of reads map. In serum exosome samples, about 55% of reads mapping to hg38 genome were derived from protein coding genes; whereas 35% of tissue RNA reads mapped mainly to protein-coding transcripts (see Figure 2).Furthermore, of the approximately 12,600 detectable genes in serum samples, about 11,000 each accounted for < 0.01% of the total reads. By contrast, tissue samples showed about 22,800 genes each accounted for < 0.01% of the reads present in the ~23,500 detectable genes (see Figure 3). Detectable protein coding transcripts in each sample type (Tissue or Serum) were sorted by the average RPKM value. The top 100 transcripts were categorized by GO Term using GOTermMapper; specifically, GOA GO Slim Database and Biological Process ontology (see Figure 4).

To learn more about ORB’s in silico service offerings for biofluid-based exosomal mRNA sequencing data, please visit our  long RNA sequencing bioinfomatics page!

 
Fig. 2 Gene Abundance Distribution.
Fig. 3 Gene Ontology Categories: top 100 protein coding categories.


Related Services

ORB's biofluid exosomal long RNA profiling services can support biomarker discovery and companion diagnostics initiatives through the identification of reliable markers.  ORB computational biologists utilize a consultative approach to ensure the most appropriate statistical analysis are utilized in predictive modeling services in order to select gene candidates that best align with each project's unique research goals.  To support FDA and patent applications, ORB provides GLP certified real-time quantitative PCR assays to confirm initial findings from RNA sequencing studies.  Follow the links below for more information about these related services.


Contact us to learn more about ORB's exosomal long RNA profiling service and how these analyses can advance your research!

References

  1. Raposo, G., & Stoorvogel, W. (2013). Extracellular vesicles: Exosomes, microvesicles, and friends. The Journal of Cell Biology, 200(4), 373-383. doi:10.1083/jcb.201211138
  2. Kosaka, N., Iguchi, H., & Ochiya, T. (2010). Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis. Cancer science101(10), 2087-2092.
  3. Izzotti A, Carozzo S, Pulliero A, Zhabayeva D, Ravetti JL, Bersimbaev R. Extracellular MicroRNA in liquid biopsy: applicability in cancer diagnosis and prevention. Am J Cancer Res. 2016 Jul 1;6(7):1461-93.
  4. Yang, J., Wei, F., Schafer, C., & Wong, D. T. (2014). Detection of tumor cell-specific mRNA and protein in exosome-like microvesicles from blood and saliva. PloS one9(11), e110641.
  5. Lee, Y., Andaloussi, S. E., & Wood, M. J. (2012). Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy. Human molecular genetics21(R1), R125-R134.
  6. Huang, X., Yuan, T., Tschannen, M., Sun, Z., Jacob, H., Du, M., & Kohli, M. (2013). Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC genomics14(1), 319.
  7. Lin, J., Li, J., Huang, B., Liu, J., Chen, X., Chen, X. M., ... & Wang, X. Z. (2015). Exosomes: novel biomarkers for clinical diagnosis. The scientific world journal2015.
  8. Nilsson, J., Skog, J., Nordstrand, A., Baranov, V., Mincheva-Nilsson, L., Breakefield, X. O., & Widmark, A. (2009). Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. British journal of cancer100(10), 1603-1607.
  9. Qi, P., & Du, X. (2013). The long non-coding RNAs, a new cancer diagnostic and therapeutic gold mine. Modern Pathology26(2), 155-165.
  10. Mohankumar S, Patel T. Extracellular vesicle long noncoding RNA as potential biomarkers of liver cancer. Brief Funct Genomics. 2016 May;15(3):249-56. doi: 10.1093/bfgp/elv058.