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

PLoS One. 2020 Nov 6;15(11):e0238545. doi: 10.1371/journal.pone.0238545. eCollection 2020.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / metabolism
  • Cell Line, Tumor
  • Extracellular Vesicles / genetics*
  • Extracellular Vesicles / metabolism
  • Humans
  • Membranes / metabolism*
  • RNA / genetics*
  • Ultracentrifugation / methods

Substances

  • Biomarkers
  • RNA

Grants and funding

This work was funded by Fondo Sectorial de Investigacion en Salud y Seguridad Social FOSISS Ref. No. 290432. The funding was awarded to MMAH https://www.conacyt.gob.mx/index.php/fondos-sectoriales-constituidos2/item/ssa-imss-issste-conacyt The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.