Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium.
Center for Medical Genetics, Ghent University, Ghent, Belgium.
J Extracell Vesicles. 2014 Sep 18;3. doi: 10.3402/jev.v3.24858. eCollection 2014.
Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.
尽管人们对细胞外囊泡（包括外泌体）在癌症中的作用及其作为诊断、预后、药物反应和复发的生物标志物的应用非常感兴趣，但目前仍缺乏可靠的分离方案。我们对 4 种外泌体分离方案进行了比较评估，以评估其可用性、产量和纯度，以及对下游标志物发现的组学方法的影响。OptiPrep 密度梯度离心在纯度方面优于超速离心和 ExoQuick 和 Total Exosome Isolation 沉淀，这体现在具有最多的 CD63 阳性纳米囊泡、在外泌体标记蛋白中最高的富集以及缺乏 Argonaute-2 复合物等污染蛋白上。最纯的外泌体部分揭示了一个独特的富含翻译、核糖体、线粒体和核腔功能的 mRNA 谱。我们的结果表明，实施高纯度技术是获得可靠组学数据和鉴定外泌体特异性功能和生物标志物的前提。
