纳米粒子跟踪分析
色谱法
大小排阻色谱法
细胞外小泡
胞外囊泡
微泡
生物
核糖核酸
RNA提取
分离(微生物学)
超滤(肾)
DNA
计算生物学
化学
生物化学
生物信息学
基因
小RNA
细胞生物学
酶
作者
Samaneh Sharif,Sina Mozaffari‐Jovin,Farzaneh Alizadeh,Majid Mojarrad,Hossein Baharvand,Maryam Nouri,Manouchehr Vaziri
标识
DOI:10.1016/j.jddst.2023.104796
摘要
Small extracellular vesicles (sEVs) are nano-scale particles that can be separated from various types of biofluids. They carry DNA, RNA, and protein materials that vary by the type of releasing cells and thus can serve as novel disease biomarkers. However, the heterogeneity of isolated exosome populations, as one of the main types of sEVs, as well as their contamination with impurities makes it difficult to precisely study their molecular composition and biogenesis. To overcome this limitation, pure sEVs must be prepared. However, isolation of pure sEVs, particularly from blood samples, is currently challenging and the use of conventional methods often leads to the co-purification of plasma soluble proteins and lipoproteins in the final product. Here, we provide a guideline for the isolation of high-purity sEVs from blood plasma based on size exclusion chromatography (SEC) using a homemade sephacryl S-400 column. SEC combined with proteinase K pre-treatment and ultrafiltration of plasma samples led to significant removal of contaminating plasma proteins in purified sEVs. Using this approach, two subpopulations of sEVs were reproducibly isolated from blood samples of colorectal cancer (CRC) patients. Small amounts of plasma samples are required to isolate a wide range of sEVs, such as Exo-S and Exo-L. Furthermore, the method was optimized for the extraction of DNA and RNA from sEVs for diagnostic purposes. The development of this simple and easy-to-use SEC method with minimal protein contaminants paves the way for translational studies of subpopulations of sEVs for diagnostic, prognostic, and therapeutic applications.
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