JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Published: June 14th, 2013

DOI:

10.3791/50294

1Department of Pharmacology & Physiology, Drexel University College of Medicine

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.

Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.

These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.

Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.

These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

Short noncoding miRNAs modulate gene expression by binding to the target mRNA. Seed sequence complementarity of ~7 base pairs enables miRNA to bind to the target mRNA resulting in the inhibition of translation or in reduction in the stability of the mRNA, both of which can result in decreased expression of the target protein 1. Research over the last decade has unequivocally proven a fundamental role for miRNAs in mediating cellular functions. There has also been considerable effort directed towards dissecting miRNA mediated molecular changes underlying various diseases 2,3. Furthermore, recent identification of stable miRNAs in bodily fluids

Log in or to access full content. Learn more about your institution’s access to JoVE content here

All experiments using blood samples from human and rodents were executed in compliance with all relevant guidelines, regulations and regulatory agencies. Human subjects were enrolled after giving informed consent as approved by the Drexel University College of Medicine Institutional Review Board and all procedures for studies performed using animals were approved by Drexel's Institutional Animal Care and Use Committee.

1. Exosome Purification from Blood (~5.5 hr)

TIPS

Log in or to access full content. Learn more about your institution’s access to JoVE content here

After isolating exosomes from blood or cell culture media, the purity of the exosomes can be tested by electron microscopy (EM) and western blot (Figures 1A and 1B). We confirmed our exosome preparations from various sources with EM and western blot using multiple antibodies. Figure 1A shows EM images confirming that exosomes are intact with a diameter of ~30 -100 nm and contain CD81 by immunogold labeling. Commonly used exosomal markers are Hsp70 and tetraspannin family.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

In this protocol, we show the quantification of miRNAs and mRNAs from exosomes purified by differential centrifugation from blood and culture media. Exosomes have diverse components dependent upon their origin and are involved in a number of biological functions, including immune response, antigen presentation, intracellular communication, and the transfer of RNA and proteins 9,11,12,19,20. While size and shape is a determinant of exosome purity, a number of papers showing EM data of exosomes indicate that the.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This study was supported by funds from Rita Allen Foundation grant to Seena Ajit. The authors would like to acknowledge Erika Balogh and Dr. Soumitra Ghoshroy from the University of South Carolina Electron Microscopy Center for instrument use, scientific and technical assistance.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Name of Reagent/Material Company Catalog Number Comments
EDTA coated vacutainer (10 ml tubes) BD Diagnostics 366643 For exosome purification from human blood
EDTA coated vacutainer (2 ml tubes) BD Diagnostics 367841 For exosome purification from mouse blood
PAXgene Blood RNA Tube BD Diagnostics 762165 For miRNA isolation from total blood
miRVana microRNA isolation kit Ambion AM1561
Acid-Phenol: CHCl3 Ambion 9721G
DNase 1 Qiagen 79254
TaqMan Universal PCR Master Mix, No AmpErase UNG Applied Biosystems 4326614 For TLDA cards
Megaplex RT rodent Pool Set v3.0 Applied Biosystems 4444746
Megaplex preamp rodent Pool set v3.0 Applied Biosystems 4444747
Taqman Array Rodent MicroRNA A+B set V3.0 Applied Biosystems 4444909
Megaplex RT Human Pool set V3.0 Applied Biosystems 4444745
Megaplex Preamp human pool set v3.0 Applied Biosystems 4444748
Taqman Array Human MicroRNA A+B Cards Set v3.0 Applied Biosystems 4444913
Taqman MicroRNA RT kit Applied Biosystems 4366596
Taqman fast universal PCR master mix Applied Biosystems 4366072 For mRNA qRT-PCR
Tumor necrosis factor (primer probe) Applied Biosystems Hs01113624_g1
Vascular endothelial growth factor A (primer probe) Applied Biosystems Hs00900055_m1
Maxima First Strand cDNA Synthesis Kit for RT-qPCR Thermo Scientific K1642 For mRNA
HSP70 antibody Abcam ab94368 For western blot
Anti-rabbit IgG-Gold Sigma G7402 For electron microscopy
Rabbit-anti CD81 Sigma SAB3500454
Nickel 300 mesh carbon formvar grids Electron Microscopy Sciences FCF300-Ni
Copper 300 mesh carbon formvar grids Electron Microscopy Sciences FCF300-Cu
Table 1. Table of specific reagents.

  1. Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell. 136, 215-233 (2009).
  2. Mendell, J. T., Olson, E. N. MicroRNAs in stress signaling and human disease. Cell. 148, 1172-1187 (2012).
  3. Esteller, M. Non-coding RNAs in human disease. Nature reviews. Genetics. 12, 861-874 (2011).
  4. Mitchell, P. S., et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proceedings of the National Academy of Sciences of the United States of America. 105, 10513-10518 (2008).
  5. Chen, X., et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell research. 18, 997-1006 (2008).
  6. Gilad, S., et al. Serum microRNAs are promising novel biomarkers. PloS one. 3, e3148 (2008).
  7. Mittelbrunn, M., et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nature. 2, 282 (2011).
  8. Valadi, H., et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 9, 654-659 (2007).
  9. Gyorgy, B., et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci. 68, 2667-2688 (2011).
  10. Mathivanan, S., Ji, H., Simpson, R. J. Exosomes: extracellular organelles important in intercellular communication. J. Proteomics. 73, 1907-1920 (2010).
  11. Ramachandran, S., Palanisamy, V. Horizontal transfer of RNAs: exosomes as mediators of intercellular communication. Wiley Interdiscip Rev. RNA. , (2011).
  12. Record, M., Subra, C., Silvente-Poirot, S., Poirot, M. Exosomes as intercellular signalosomes and pharmacological effectors. Biochem Pharmacol. 81, 1171-1182 (2011).
  13. Thery, C., Ostrowski, M., Segura, E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 9, 581-593 (2009).
  14. Ludwig, A. K., Giebel, B. Exosomes: small vesicles participating in intercellular communication. The international journal of biochemistry & cell biology. 44, 11-15 (2012).
  15. Thery, C., Amigorena, S., Raposo, G., Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Chapter 3, Unit 3 22 (2006).
  16. Masyuk, A. I., et al. Biliary exosomes influence cholangiocyte regulatory mechanisms and proliferation through interaction with primary cilia. American journal of physiology. Gastrointestinal and liver physiology. 299, G990-G999 (2010).
  17. Fauré, J., et al. Exosomes are released by cultured cortical neurones. Molecular and Cellular Neuroscience. 31, 642-648 (2006).
  18. Waldenstrom, A., Genneback, N., Hellman, U., Ronquist, G. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells. PloS one. 7, e34653 (2012).
  19. Simpson, R. J., Lim, J. W., Moritz, R. L., Mathivanan, S. Exosomes: proteomic insights and diagnostic potential. Expert Rev Proteomics. 6, 267-283 (2009).
  20. Turchinovich, A., Weiz, L., Langheinz, A., Burwinkel, B. Characterization of extracellular circulating microRNA. Nucleic acids research. 39, 7223-7233 (2011).
  21. El-Andaloussi, S., et al. Exosome-mediated delivery of siRNA in vitro and in vivo. Nature. 7, 2112-2126 (2012).
  22. Singh, P. P., Smith, V. L., Karakousis, P. C., Schorey, J. S. Exosomes isolated from mycobacteria-infected mice or cultured macrophages can recruit and activate immune cells in vitro and in vivo. J. Immunol. 189, 777-785 (2012).
  23. Etheridge, A., Lee, I., Hood, L., Galas, D., Wang, K. Extracellular microRNA: A new source of biomarkers. Mutat. Res. , (2011).
  24. Stenvang, J., Silahtaroglu, A. N., Lindow, M., Elmen, J., Kauppinen, S. The utility of LNA in microRNA-based cancer diagnostics and therapeutics. Seminars in cancer biology. 18, 89-102 (2008).
  25. Mathivanan, S., Fahner, C. J., Reid, G. E., Simpson, R. J. ExoCarta 2012: database of exosomal proteins, RNA and lipids. Nucleic acids research. 40, D1241-D1244 (2012).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved