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Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs.
J Extracell Vesicles. 2019; 8(1):1555410.JE

Abstract

The microRNA (miRNA) cargo contained in plasma extracellular vesicles (EVs) offers a relatively little explored source of biomarkers for brain diseases that can be obtained noninvasively. Methods to isolate EVs from plasma, however, are still being developed. For EV isolation, it is important to ensure the removal of vesicle-free miRNAs, which account for approximately two-thirds of plasma miRNAs. Membrane particle precipitation-based EV isolation is an appealing method because of the simple protocol and high yield. Here, we evaluated the performance of a precipitation-based method to obtain enriched EV-specific miRNAs from a small volume of rat plasma. We performed size-exclusion chromatography (SEC) on precipitation-isolated EV pellets and whole plasma. The SEC fractions were analysed using Nanoparticle Tracking Analysis (NTA), protein and miRNA concentration assays, and droplet digital polymerase chain reaction for four miRNAs (miR-142-3p, miR-124-3p, miR-23a, miR-122). Precipitation-isolated EVs and selected SEC fractions from the plasma were also analysed with transmission electron microscopy (TEM). Precipitation-based EV isolation co-precipitated 9% to 15% of plasma proteins and 21% to 99% of vesicle-free miRNAs, depending on the individual miRNAs. In addition, the amount of miR-142-3p, found mainly in EV fractions, was decreased in the EV fractions, indicating that part of it was lost during precipitation-based isolation. Western blot and TEM revealed both protein and lipoprotein contamination in the precipitation-isolated EV-pellets. Our findings indicate that a precipitation-based method is not sufficient for purifying plasma EV-contained miRNA cargo. The particle number measured by NTA is high, but this is mostly due to the contaminating lipoproteins. Although a part of the vesicle-free miRNA is removed, vesicle-free miRNA still dominates in plasma EV pellets isolated by the precipitation-based method.

Links

Authors+Show Affiliations

A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

Navarro-Ferrandis V

A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.

SIB Labs, University of Eastern Finland, Kuopio, Finland.

A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

30574280

Citation

Karttunen, Jenni, et al. "Precipitation-based Extracellular Vesicle Isolation From Rat Plasma Co-precipitate Vesicle-free MicroRNAs." Journal of Extracellular Vesicles, vol. 8, no. 1, 2019, p. 1555410.

Karttunen J, Heiskanen M, Navarro-Ferrandis V, et al. Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs. J Extracell Vesicles. 2019;8(1):1555410.

Karttunen, J., Heiskanen, M., Navarro-Ferrandis, V., Das Gupta, S., Lipponen, A., Puhakka, N., Rilla, K., Koistinen, A., & Pitkänen, A. (2019). Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs. Journal of Extracellular Vesicles, 8(1), 1555410. https://doi.org/10.1080/20013078.2018.1555410

Karttunen J, et al. Precipitation-based Extracellular Vesicle Isolation From Rat Plasma Co-precipitate Vesicle-free MicroRNAs. J Extracell Vesicles. 2019;8(1):1555410. PubMed PMID: 30574280.

* Article titles in AMA citation format should be in sentence-case

TY - JOUR T1 - Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs. AU - Karttunen,Jenni, AU - Heiskanen,Mette, AU - Navarro-Ferrandis,Vicente, AU - Das Gupta,Shalini, AU - Lipponen,Anssi, AU - Puhakka,Noora, AU - Rilla,Kirsi, AU - Koistinen,Arto, AU - Pitkänen,Asla, Y1 - 2018/12/18/ PY - 2018/04/10/received PY - 2018/11/21/revised PY - 2018/11/23/accepted PY - 2018/12/22/entrez PY - 2018/12/24/pubmed PY - 2018/12/24/medline KW - Extracellular vesicle KW - ddPCR KW - extracellular vesicle isolation KW - miRNA KW - plasma KW - precipitation KW - size-exclusion chromatography SP - 1555410 EP - 1555410 JF - Journal of extracellular vesicles JO - J Extracell Vesicles VL - 8 IS - 1 N2 - The microRNA (miRNA) cargo contained in plasma extracellular vesicles (EVs) offers a relatively little explored source of biomarkers for brain diseases that can be obtained noninvasively. Methods to isolate EVs from plasma, however, are still being developed. For EV isolation, it is important to ensure the removal of vesicle-free miRNAs, which account for approximately two-thirds of plasma miRNAs. Membrane particle precipitation-based EV isolation is an appealing method because of the simple protocol and high yield. Here, we evaluated the performance of a precipitation-based method to obtain enriched EV-specific miRNAs from a small volume of rat plasma. We performed size-exclusion chromatography (SEC) on precipitation-isolated EV pellets and whole plasma. The SEC fractions were analysed using Nanoparticle Tracking Analysis (NTA), protein and miRNA concentration assays, and droplet digital polymerase chain reaction for four miRNAs (miR-142-3p, miR-124-3p, miR-23a, miR-122). Precipitation-isolated EVs and selected SEC fractions from the plasma were also analysed with transmission electron microscopy (TEM). Precipitation-based EV isolation co-precipitated 9% to 15% of plasma proteins and 21% to 99% of vesicle-free miRNAs, depending on the individual miRNAs. In addition, the amount of miR-142-3p, found mainly in EV fractions, was decreased in the EV fractions, indicating that part of it was lost during precipitation-based isolation. Western blot and TEM revealed both protein and lipoprotein contamination in the precipitation-isolated EV-pellets. Our findings indicate that a precipitation-based method is not sufficient for purifying plasma EV-contained miRNA cargo. The particle number measured by NTA is high, but this is mostly due to the contaminating lipoproteins. Although a part of the vesicle-free miRNA is removed, vesicle-free miRNA still dominates in plasma EV pellets isolated by the precipitation-based method. SN - 2001-3078 UR - https://www.unboundmedicine.com/medline/citation/30574280/Precipitation_based_extracellular_vesicle_isolation_from_rat_plasma_co_precipitate_vesicle_free_microRNAs_ DB - PRIME DP - Unbound Medicine ER -