Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation.

Gilberto Gutiérrez García,Maria Montserrat Aguilar-Hernandez,Gabriela Galicia García,Álvaro Aguayo,Andrea Izquierdo Medina,Cynthia Paola López Pacheco,Mariana Rotzinger-Rodríguez,Gustavo Antonio Casas Aguilar,Jessica Zalapa Soto,Ilja Kusters

doi:10.1371/journal.pone.0238545

Abstract

Extracellular vesicles (EV) have attracted much attention as potential biomarkers due to their protein, RNA and other nucleic acid content. The most common method used for EV isolation is differential ultracentrifugation (DU), however given the DU technical difficulties, other more practical methods have surged, such as membrane-affinity column commercial kits. Here, we assessed one commercial kit in terms of EV recovery and EV-derived RNA yield and compared it with a DU protocol. Our data shows that the commercial kit preparation results in a lower count of EV-like structures and a reduced expression of EV markers when compared to DU samples. Thus, apparently suggesting that the commercial kit had a lower EV yield. However, these findings did not reflect on RNA yield, which was greater with the commercial kit, even after an enzymatic treatment with proteinase K and RNAse A. We conclude that the kit has a higher EV-derived RNA yield in comparison to our DU protocol, suggesting that it may be the method of choice for RNA sequencing purposes.

Highlights

Extracellular vesicles (EV) is a generic term used to define particles delimited by a lipid bilayer that originate from the cell [1,2]
EV were isolated from culture supernatant by differential ultracentrifugation (DU) and ExoEasy kit We investigated the presence of EV in DU-CS and Exo-CS samples by electron microscopy
We compared the characteristics of ExoEasy kit derived EV as well as their RNA content to those obtained by DU when processing culture supernatant and plasma samples

Summary

Introduction

Extracellular vesicles (EV) is a generic term used to define particles delimited by a lipid bilayer that originate from the cell [1,2]. Classification of EV has been a source of debate, since many definitions have been proposed. One of the earliest and most used nomenclatures divided EV into two main subtypes: microvesicles and exosomes [3,4]. They were differentiated by size and mechanism of biogenesis. MISEV 2018 newer consensus has proposed that EV definitions should be based more on the size and biophysical properties of EV, since it is difficult to determine the biological origin of vesicles [1]

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Results

Conclusion