Skip to main content
SpringerLink
Log in

Isolation, Extraction and Deep-Sequencing Analysis of Extracellular RNAs (exRNAs) from Human Plasma

  • Protocol
  • First Online:
Small Non-Coding RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2300))

Abstract

Extracellular RNAs (exRNAs) are secreted by nearly all cell types and are now known to play multiple physiological roles. Human plasma, a readily available sample for biomedical analysis, was reported to contain various subpopulations of exRNA, some of which are most likely components of plasma ribonucleoproteins (RNPs), while others are encapsulated into extracellular vesicles (EVs) of different size, origin, and composition. Unbiased analysis of exRNA composition can be performed with prefractionation of plasma exRNA followed by library preparation, sequencing, and bioinformatics analysis. In addition to “mature,” adaptor ligation-competent RNA species (5′-P/3′-OH), human plasma contains a substantial proportion of degraded RNA fragments, featuring 5′-OH/3′-P or cyclophosphate extremities, which can be made competent for ligation using appropriate treatment. Polyethylene glycol (PEG)-based precipitation kits for EV isolation yield a fraction that is highly contaminated by large RNPs and EV-associated RNAs. Purer EV preparations are obtained by using Proteinase K and RNase A treatment, as well as by size-exclusion chromatography (SEC).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Sadik N, Cruz L, Gurtner A, Rodosthenous RS, Dusoswa SA, Ziegler O, Van Solinge TS, Wei Z, Salvador-Garicano AM, Gyorgy B et al (2018) Extracellular RNAs: a new awareness of old perspectives. Methods Mol Biol 1740:1–15

    Article  CAS  Google Scholar 

  2. Li K, Rodosthenous RS, Kashanchi F, Gingeras T, Gould SJ, Kuo LS, Kurre P, Lee H, Leonard JN, Liu H et al (2018) Advances, challenges, and opportunities in extracellular RNA biology: insights from the NIH exRNA strategic workshop. JCI Insight 3:e98942

    Article  Google Scholar 

  3. Yeri A, Courtright A, Reiman R, Carlson E, Beecroft T, Janss A, Siniard A, Richholt R, Balak C, Rozowsky J et al (2017) Total extracellular Small RNA profiles from plasma, saliva, and urine of healthy subjects. Sci Rep 7:44061

    Article  CAS  Google Scholar 

  4. Griswold AJ, Perez J, Nuytemans K, Strong TA, Wang L, Vance DD, Ennis H, Smith MK, Best TM, Vance JM et al (2018) Transcriptomic analysis of synovial extracellular RNA following knee trauma: a pilot study. J Orthop Res 36:1659–1665

    Article  CAS  Google Scholar 

  5. Lässer C, Seyed Alikhani V, Ekström K, Eldh M, Torregrosa Paredes P, Bossios A, Sjöstrand M, Gabrielsson S, Lötvall J, Valadi H (2011) Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med 9:9

    Article  Google Scholar 

  6. Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL et al (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 108:5003–5008

    Article  CAS  Google Scholar 

  7. Turchinovich A, Weiz L, Langheinz A, Burwinkel B (2011) Characterization of extracellular circulating microRNA. Nucleic Acids Res 39:7223–7233

    Article  CAS  Google Scholar 

  8. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:423–433

    Article  CAS  Google Scholar 

  9. Zhang H, Freitas D, Kim HS, Fabijanic K, Li Z, Chen H, Mark MT, Molina H, Martin AB, Bojmar L et al (2018) Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat Cell Biol 20:332–343

    Article  CAS  Google Scholar 

  10. György B, Szabó TG, Pásztói M, Pál Z, Misják P, Aradi B, László V, Pállinger É, Pap E, Kittel Á et al (2011) Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 68:2667–2688

    Article  Google Scholar 

  11. Momen-Heravi F, Getting SJ, Moschos SA (2018) Extracellular vesicles and their nucleic acids for biomarker discovery. Pharmacol Ther 192:170–187

    Article  CAS  Google Scholar 

  12. Greening DW, Simpson RJ (2018) Understanding extracellular vesicle diversity - current status. Expert Rev Proteomics 15:887–910

    Article  CAS  Google Scholar 

  13. Zaborowski MP, Balaj L, Breakefield XO, Lai CP (2015) Extracellular vesicles: composition, biological relevance, and methods of study. Bioscience 65:783–797

    Article  Google Scholar 

  14. Crescitelli R, Lässer C, Szabó TG, Kittel A, Eldh M, Dianzani I, Buzás EI, Lötvall J (2013) Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J Extracell Vesicles 2

    Google Scholar 

  15. Savelyeva AV, Kuligina EV, Bariakin DN, Kozlov VV, Ryabchikova EI, Richter VA, Semenov DV (2017) Variety of RNAs in peripheral blood cells, plasma, and plasma fractions. Biomed Res Int 2017:7404912

    Article  Google Scholar 

  16. Mitchell AJ, Gray WD, Hayek SS, Ko Y-A, Thomas S, Rooney K, Awad M, Roback JD, Quyyumi A, Searles CD (2016) Platelets confound the measurement of extracellular miRNA in archived plasma. Sci Rep 6:32651

    Article  CAS  Google Scholar 

  17. Mateescu B, Kowal EJK, van Balkom BWM, Bartel S, Bhattacharyya SN, Buzás EI, Buck AH, de Candia P, Chow FWN, Das S et al (2017) Obstacles and opportunities in the functional analysis of extracellular vesicle RNA—an ISEV position paper. J Extracell Vesicles 6:1286095

    Article  Google Scholar 

  18. Eldh M, Lötvall J, Malmhäll C, Ekström K (2012) Importance of RNA isolation methods for analysis of exosomal RNA: evaluation of different methods. Mol Immunol 50:278–286

    Article  CAS  Google Scholar 

  19. Hoy AM, Buck AH (2012) Extracellular small RNAs: what, where, why?: figure 1. Biochem Soc Trans 40:886–890

    Article  CAS  Google Scholar 

  20. Etheridge A, Wang K, Baxter D, Galas D (2018) Preparation of Small RNA NGS libraries from biofluids. Methods Mol Biol 1740:163–175

    Article  CAS  Google Scholar 

  21. Soekmadji C, Hill AF, Wauben MH, Buzás EI, Di Vizio D, Gardiner C, Lötvall J, Sahoo S, Witwer KW (2018) Towards mechanisms and standardization in extracellular vesicle and extracellular RNA studies: results of a worldwide survey. J Extracell Vesicles 7:1535745

    Article  CAS  Google Scholar 

  22. Small J, Roy S, Alexander R, Balaj L (2018) Overview of protocols for studying extracellular RNA and extracellular vesicles. Methods Mol Biol 1740:17–21

    Article  CAS  Google Scholar 

  23. Théry C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3: Unit 3.22

    Google Scholar 

  24. Colhoun HM, Otvos JD, Rubens MB, Taskinen MR, Underwood SR, Fuller JH (2002) Lipoprotein subclasses and particle sizes and their relationship with coronary artery calcification in men and women with and without type 1 diabetes. Diabetes 51:1949–1956

    Article  CAS  Google Scholar 

  25. Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A (2014) The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles 3

    Google Scholar 

  26. Andreu Z, Rivas E, Sanguino-Pascual A, Lamana A, Marazuela M, González-Alvaro I, Sánchez-Madrid F, de la Fuente H, Yáñez-Mó M (2016) Comparative analysis of EV isolation procedures for miRNAs detection in serum samples. J Extracell Vesicles 5:31655

    Article  Google Scholar 

  27. Stranska R, Gysbrechts L, Wouters J, Vermeersch P, Bloch K, Dierickx D, Andrei G, Snoeck R (2018) Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma. J Transl Med 16:1

    Article  CAS  Google Scholar 

  28. Böing AN, van der Pol E, Grootemaat AE, Coumans FAW, Sturk A, Nieuwland R (2014) Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles 3

    Google Scholar 

  29. Galvanin A, Dostert G, Ayadi L, Marchand V, Velot É, Motorin Y (2019) Diversity and heterogeneity of extracellular RNA in human plasma. Biochimie 164:22–36

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to the EFS (Etablissement Français du Sang) for providing us blood samples used for our research. This work was co-funded by a grant from the Université de Lorraine and from the European Union in the framework of the FEDER-FSE program “Lorraine et Massif des Vosges 2014-2020.”

Author information

Authors and Affiliations

  1. Université de Lorraine, CNRS, INSERM, IBSLOR, F-54000 Nancy, France

    Virginie Marchand & Yuri Motorin

  2. Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France

    Adeline Galvanin & Yuri Motorin

Authors
  1. Virginie Marchand
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adeline Galvanin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuri Motorin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Virginie Marchand .

Editor information

Editors and Affiliations

  1. Université de Lorraine, CNRS, IMoPA, Nancy, France

    Mathieu Rederstorff

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Marchand, V., Galvanin, A., Motorin, Y. (2021). Isolation, Extraction and Deep-Sequencing Analysis of Extracellular RNAs (exRNAs) from Human Plasma. In: Rederstorff, M. (eds) Small Non-Coding RNAs. Methods in Molecular Biology, vol 2300. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1386-3_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1386-3_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1385-6

  • Online ISBN: 978-1-0716-1386-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation