Yang Yang1, Yaojie Wang2, Sisi Wei2, Chaoxi Zhou1, Jiarui Yu3, Guiying Wang1, Wenxi Wang4, Lianmei Zhao5. 1. Department of General Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050011, Hebei, China. 2. Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050011, Hebei, China. 3. Department of Radiation Oncology, North China University of Science and Technology Affiliated People's Hospital, Tangshan, 063000, Hebei, China. 4. Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050011, Hebei, China. wenxi0316@163.com. 5. Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050011, Hebei, China. zhaolianmei@hbydsy.com.
Abstract
BACKGROUND: Extracellular vesicles (EVs), known as cell-derived membranous structures harboring a variety of biomolecules, have been widely used in liquid biopsy. Due to the complex biological composition of plasma, plasma RNA omics analysis (RNomics) is easily affected, thus it is necessary to select an optimal strategy from exiting methods according to the performance for intended application. METHODS: In this study, four different strategies for EVs isolation were performed and compared (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy). We compared the yield, purity, PCR quantification of RNAs, miRNA-seq analyses and mRNA-seq analyses of RNAs from EVs isolated using four methods. RESULTS: The results showed that the lowest miRNA binding protein AGO2 (Argonaute-2) and the highest EVs-specific miRNA and lncRNA were observed in EVs obtained through SEC, meanwhile the content of the non-specific miRNA was the lowest. Further RNA-Seq data revealed that RNAs obtained via SEC presented more useful reads for both miRNA and mRNA. Furthermore, the mRNA delivered via SEC tended to have a concentration comparable to the ideal FPKM (Fragments Per Kilobase Million) value. CONCLUSIONS: SEC shall be used as an optimal strategy for the isolation of EVs in plasma RNomics analysis.
BACKGROUND: Extracellular vesicles (EVs), known as cell-derived membranous structures harboring a variety of biomolecules, have been widely used in liquid biopsy. Due to the complex biological composition of plasma, plasma RNA omics analysis (RNomics) is easily affected, thus it is necessary to select an optimal strategy from exiting methods according to the performance for intended application. METHODS: In this study, four different strategies for EVs isolation were performed and compared (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy). We compared the yield, purity, PCR quantification of RNAs, miRNA-seq analyses and mRNA-seq analyses of RNAs from EVs isolated using four methods. RESULTS: The results showed that the lowest miRNA binding protein AGO2 (Argonaute-2) and the highest EVs-specific miRNA and lncRNA were observed in EVs obtained through SEC, meanwhile the content of the non-specific miRNA was the lowest. Further RNA-Seq data revealed that RNAs obtained via SEC presented more useful reads for both miRNA and mRNA. Furthermore, the mRNA delivered via SEC tended to have a concentration comparable to the ideal FPKM (Fragments Per Kilobase Million) value. CONCLUSIONS: SEC shall be used as an optimal strategy for the isolation of EVs in plasma RNomics analysis.
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