Comparision of serum exosomes and exosomal RNA isolation methods / 中华检验医学杂志

ABSTRACT

Objective The effect of serum exosome isolation method on obtained exosomal RNA is not yet clear .The aim of this study was to provide evidences to selected exosome isolation methods .Methods This was a method comparison study to assess three methods .Vein blood samples were collected from 3 healthy donors from Nanfang hospital , 4 ℃ 12 h was taken to make blood natural coagulated and the supernatant was taken as the serum . Exosomes extracted by Ultracentrifugation , ExoQuick and Total Exosome Isolation Reagent ( TEI ) kits from serum were assessed by transmission electron microscopy , nanoparticle tracking analysis and protein analysis to identify morphology , protein expression , concentration and size distribution of particles .ExoRNA extracted by Trizol-LS, SeraMir, Total Exosome RNA Isolation ( TER) and exoRNeasy were assessed by Bioanalyzer 2100 and qPCR to ascertain RNA distribution and miRNA expression.Results For exosomes isolation , two commercial kits ( ExoQuick and TEI ) showed higher exosomes recovery and protein concentration than ultracentrifugation , but exosome isolated by ultracentrifugation expressed abundant protein makers mostly .For exo-RNA isolation, the distribution of RNA and expression of miRNAinfluenced by three methods , but there was no effect on the relative expression trend of miRNA.ExoRNeasy resulted in the highest yield and most narrow size distribution pattern of small RNA with higher level of miRNA expression .Results of TEI with TER kits showed no obvious bands of small RNA and moderate miRNA expression among methods .Ultra with Trizol-LS or ExoQuick with SeraMir showed low concentration measurable bands around 100 nt, with changeable miRNA profiling irregularly . Conclusions Extraction of exosomes using traditional ultracentrifugation method is applicable for proteomics research work .Extraction using ExoQuick and TEI kits is suitable for preparing exosomes from scarce samples such as serum.