Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers
Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers
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DOI：
10.1038/ki.2012.256
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发表时间：
2012-11-01
影响因子：
19.6
通讯作者：
DiStefano, Johanna K.
中科院分区：
文献类型：
--
作者：
Alvarez, M. Lucrecia;Khosroheidari, Mahdieh;DiStefano, Johanna K.
尿外泌体是40- 100 nm的囊泡，含有蛋白质、mRNA和microRNA，可作为肾功能障碍和结构损伤的生物标志物。目前，需要更敏感和特异的肾损伤和疾病进展的生物标志物。在这里，我们试图确定用于蛋白质组学分析和RNA分析的最佳外泌体分离方法，作为生物标志物发现的第一步。我们使用了六种不同的方案;三种基于超离心，一种使用基于纳米膜浓缩器的方法，两种使用商业外泌体沉淀试剂。使用改良的外泌体沉淀方案获得外泌体的最高产量，其也产生最高量的微小RNA和mRNA，因此对于随后的RNA分析是理想的。如果无法获得超浓缩物和/或需要处理大量样品，该方法同样适用于下游蛋白质组学分析。然而，如果超离心是可用的，并且很少的样品将被处理用于蛋白质组学分析，则两种超离心方法是外泌体分离的更好选择。因此，我们改进的外泌体沉淀方法是用于分离外泌体的简单、快速、高度可扩展且有效的替代方案，并且可以促进从尿液中鉴定外泌体生物标志物。Kidney International（2012）82，1024-1032; doi：10.1038/ki.2012.256; 2012年7月11日在线发表
Urinary exosomes are 40-100nm vesicles containing protein, mRNA, and microRNA that may serve as biomarkers of renal dysfunction and structural injury. Currently, there is a need for more sensitive and specific biomarkers of renal injury and disease progression. Here we sought to identify the best exosome isolation methods for both proteomic analysis and RNA profiling as a first step for biomarker discovery. We used six different protocols; three were based on ultracentrifugation, one used a nanomembrane concentrator-based approach, and two utilized a commercial exosome precipitation reagent. The highest yield of exosomes was obtained using a modified exosome precipitation protocol, which also yielded the highest quantities of microRNA and mRNA and, therefore, is ideal for subsequent RNA profiling. This method is likewise suitable for downstream proteomic analyses if an ultracentrifuge is not available and/or a large number of samples are to be processed. Two of the ultracentrifugation methods, however, are better options for exosome isolation if an ultracentrifuge is available and few samples will be processed for proteomic analysis. Thus, our modified exosome precipitation method is a simple, fast, highly scalable, and effective alternative for the isolation of exosomes, and may facilitate the identification of exosomal biomarkers from urine. Kidney International (2012) 82, 1024-1032; doi:10.1038/ki.2012.256; published online 11 July 2012