Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling

Abstract

Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. Here, we analyzed the advantages and weaknesses of four commonly used commercial kits for exosomal miRNA profiling and their application to the sample of serum and/or plasma, respectively. NanoSight and Western blotting were conducted to evaluate the efficiency and purity of the isolated exosomes. In our conditions, the size distribution of the isolated particles was appropriate (40-150 nm), and ExoQuick™ Exosome Precipitation Solution (EXQ) generated a relatively high yield of exosomes. Nevertheless, albumin impurity was ubiquitous for all the four kits, and Total Exosome Isolation for serum or plasma (TEI) yielded a relatively pure isolation. We further performed Illumina sequencing combined with RT-qPCR to determine the ability of these kits for miRNA profiling. There was significant correlation of the exosomal miRNA profile and specific miRNAs between kits, but with differences depending on methods. exoRNeasy Serum/Plasma Midi Kit (EXR) and EXQ performed better in the specific exosomal miRNAs recovery. Intraassay CVs for specific miRNA measurement were 0.88-3.82, 1.19-3.77, 0-2.70, and 1.23-9.11% for EXR, TEI, EXQ, and RIBO™ Exosome Isolation Reagent (REI), respectively. In each kit, serum yielded a higher abundance of exosomes and exosomal miRNAs than plasma, yet with more albumin impurity. In conclusion, our data provide some valuable guidance for the methodology of disease biomarker identification of circulation exosomal miRNAs. Graphical abstract Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. In this study, we compared four commonly used commercially available kits for exosomal miRNAsextraction and analyzed the advantages and weaknesses of each kit and their application to the sample ofserum and/or plasma.

Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives

“…During the last decade, various research groups performed studies comparing several isolation techniques in order to define the best EVs isolation workflow . To date, the most common method is differential centrifugation (DC) .…”

A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

“…The process to separate serum from blood first requires blood coagulation, which causes platelet activation and leads to increased platelet-EV secretion [61]. Sera also comprises a higher proportion of particles larger than 200 nm, compared to plasma [62] and thus plasma was selected as the starting material to optimise our biomarker workflow. SEC was chosen to isolate plasma-EVs for discovery proteomic analysis as it is rapid and reliable for isolation of small-EV subtypes from complex biological fluids.…”

Label-free detection of exosomes using a surface plasmon resonance biosensor

“…However, a major bottleneck in the development of such platforms has been the specific isolation of exosomes from complex biological fluids. Over the years, several methods utilized for the isolation of exosomes including ultracentrifugation [11], density gradient separation followed by electron microscopy [12], enzyme-linked immunosorbent assays (ELISA) [13], and western blotting [14] resulted in low purity yield, tend to be time consuming or involve extensive labelling procedures [15]. Similarly, several commercially available exosome isolation protocols or kits also co-isolate several non-exosome debris or biological material of similar physical characteristic rendering them to be ineffective.…”