Polyethylene Glycol -Mediated Exosome Isolation: A Method for Exosomal RNA Analysis

Abstract

Background: : Exosomal RNAs (ExoRNAs) offer valuable insights into their cellular origin. ExoRNA studies were faced with challenges in obtaining sufficient amounts of high-quality RNA. Herein, we aimed to compare three traditional exosome isolation methods to introduce an appropriate strategy to extract RNA from cancer-derived exosomes for further RNA analysis.

Methods: Exosomes were isolated through ultracentrifugation, precipitation kit, and size exclusion column chromatography, and then characterized by dynamic light scattering and transmission electron microscopy, followed by extracting total RNA. The quality and quantity of the extracted RNAs were assessed by a NanoDrop and 2.5% agarose gel electrophoresis.

Results: Extracted exosomes displayed a similar range of size and morphology. We found that polyethylene glycol-precipitation method resulted in a higher RNA yield with a 260/280 ratio of 1.9. The obtained exoRNA appeared as a smear in the agarose gel, indicative of small exoRNAs.

Conclusion: We provide researchers a suitable approach to isolate exosomes based on yield and purity of exoRNA.

Keywords: Exosomes; Extracellular vesicles; MicroRNA.

Fig. 1

Summary of exosomes isolation. MCF-7 cells were initially harvested, and then their supernatants were collected and purified by serial centrifugations. Afterwards, the supernatant containing the exosomes were concentrated and used for exosome isolation by SEC, PEG precipitation, and UC

Fig. 2

Size distribution analysis of exosomes isolated using (A) SEC, (B) UC, and (C) PEG precipitation methods by DLS in terms of intensity. TEM of negatively stained exosomes with a diameter ranged from 30 to 200 nm. Exosomes obtained from condition media via (D) UC, (E) PEG precipitation, and (F) SEC methods. Exosomes are shown by yellow arrows

Fig. 3

Gel electrophoresis of RNA extracted from MCF-7-derived exosomes isolated by UC, PEG precipitation, and SEC methods on a 2.5% agarose gel. Total cellular RNA was also loaded into gel as a control sample