Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs

Jenni Karttunen; Mette Heiskanen; Vicente Navarro-Ferrandis; Shalini Das Gupta; Anssi Lipponen; Noora Puhakka; Kirsi Rilla; Arto Koistinen; Asla Pitkänen

doi:10.1080/20013078.2018.1555410

Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs

J Extracell Vesicles. 2018 Dec 18;8(1):1555410. doi: 10.1080/20013078.2018.1555410. eCollection 2019.

Authors

Jenni Karttunen¹, Mette Heiskanen¹, Vicente Navarro-Ferrandis¹, Shalini Das Gupta¹, Anssi Lipponen¹, Noora Puhakka¹, Kirsi Rilla², Arto Koistinen³, Asla Pitkänen¹

Affiliations

¹ A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.
² Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
³ SIB Labs, University of Eastern Finland, Kuopio, Finland.

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.

Keywords: Extracellular vesicle; ddPCR; extracellular vesicle isolation; miRNA; plasma; precipitation; size-exclusion chromatography.

Grants and funding

This work was supported by the Academy of Finland [273909]; Academy of Finland [272249]; Sigrid Jusélius Foundation;the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement [602102 EPITARGET]; EATRIS, the European Infrastructure for Translational Medicine, University of Eastern Finland (Biocenter Kuopio) and Biocenter Finland.