Evaluation of Commercially Available Exosomal Isolation Kits from Human Plasma

Abstract

Exosomes are cell-derived lipid bilayer particles which are abundant in biological fluids.14 Exosome is an emerging source of biomarkers to diagnose various human diseases. Sequencing 15 based exosomal studies could provide a comprehensive view of exosomal RNA and protein. To16extracted these inclusions, exosomes should be isolated from the plasma first. Several exosome 17 isolation methods were introduced since the discover of exosome. To promote the clinical 18 application of exosomal inclusions, different isolation methods should be compared. We isolated 19 exosomes from human plasma by using user-friendly and commercially available kits, SBI 20 ExoQuick and QIAGEN exoRNeasy. Subsequently, small RNA sequencing was performed with two 21 groups of isolated exosome samples and one group of plasma samples. No fundamental differences 22 of exRNA yield between SC and EQ were found. In RNA profile analysis, the small RNA aligned 23 reads, miRNA pattern, sample clustering varied as a result of methodological differences. Small 24RNA isolated by ExoQuick presented better data quality and RNA profile than exoRNeasy. This 25 study compared sRNA sequencing data generated from two exosome isolation kits, it provides a 26 reference for future small RNA studies and biomarker prediction in human plasma exosome. 27 Keywords: 28 29 1. Introduction 30 Extracellular vesicles (EVs) are small lipid bilayer particles that are commonly observed in 31 human body fluids involving in cell-to-cell communication. Exosome, a major component of EVs, 32 carries the fundamental biological elements such as protein, mRNA, long non-coding RNA (lncRNA), 33 and, especially small RNA (sRNA) [1]. Recently, exosome has become an emerging source of 34 biomarkers of various human diseases like neurodegenerative diseases and cardiovascular 35 diseases[2]. Using peripheral blood to detect exosomal inclusions could satisfy the rapid and 36 noninvasive requirement of chronic disease prediction[3]. Particularly, the exosomal sRNA including 37 microRNA (miRNA) are of great diagnostic interest since they are important post-translational 38 regulators and protected by lipid bilayer structure from exposure to RNases [4]. Thus, method of 39 extracting RNA from exosomes and RNA-seq library preparation kits are developed [5-7]. Due to the 40 nature of sRNA diversity in exosomes, exosomal sRNA patterns are easily interfered, it is extremely 41 hard to reproduce the same results which have been obtained from the previous experiments [4]. A 42 few works have compared different methods of exosomal sRNA extraction by measuring isolated 43 exosome integrity and isolation efficiency from different tissues. However, previous works didn't 44 systematically analyze transcriptomic or proteomic data[8]. Our study focused on comparing next-65 QIAGEN exoRNeasy Serum/Plasma Kit, were chosen as they represent two isolation method, the66 polymer-based precipitation and the column-based chromatography, respectively. This study is 67 focusing on the impact of different exosome isola...