Department of Clinical Laboratory, Jinling Hospital, State Key Laboratory of Analytical Chemistry for Life Science, NJU Advanced Institute for Life Sciences (NAILS), Nanjing University School of Life Sciences, Nanjing University, 305 East Zhongshan Rd., Nanjing, 210002, Jiangsu, China.
State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), Nanjing University School of Life Sciences, Nanjing University, 163 Xianlin Rd., Nanjing, 210046, Jiangsu, China.
Anal Bioanal Chem. 2018 Jun;410(16):3805-3814. doi: 10.1007/s00216-018-1052-4. Epub 2018 Apr 18.
Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. Here, we analyzed the advantages and weaknesses of four commonly used commercial kits for exosomal miRNA profiling and their application to the sample of serum and/or plasma, respectively. NanoSight and Western blotting were conducted to evaluate the efficiency and purity of the isolated exosomes. In our conditions, the size distribution of the isolated particles was appropriate (40-150 nm), and ExoQuick™ Exosome Precipitation Solution (EXQ) generated a relatively high yield of exosomes. Nevertheless, albumin impurity was ubiquitous for all the four kits, and Total Exosome Isolation for serum or plasma (TEI) yielded a relatively pure isolation. We further performed Illumina sequencing combined with RT-qPCR to determine the ability of these kits for miRNA profiling. There was significant correlation of the exosomal miRNA profile and specific miRNAs between kits, but with differences depending on methods. exoRNeasy Serum/Plasma Midi Kit (EXR) and EXQ performed better in the specific exosomal miRNAs recovery. Intraassay CVs for specific miRNA measurement were 0.88-3.82, 1.19-3.77, 0-2.70, and 1.23-9.11% for EXR, TEI, EXQ, and RIBO™ Exosome Isolation Reagent (REI), respectively. In each kit, serum yielded a higher abundance of exosomes and exosomal miRNAs than plasma, yet with more albumin impurity. In conclusion, our data provide some valuable guidance for the methodology of disease biomarker identification of circulation exosomal miRNAs. Graphical abstract Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. In this study, we compared four commonly used commercially available kits for exosomal miRNAsextraction and analyzed the advantages and weaknesses of each kit and their application to the sample ofserum and/or plasma.
循环细胞外体 microRNAs（miRNAs）是有价值的生物标志物候选物；然而，关于循环细胞外体 miRNA 分析的商业试剂盒的特征和相互一致性的信息却很少。在这里，我们分析了四种常用的商业试剂盒用于外泌体 miRNA 分析的优缺点，并分别分析了它们在血清和/或血浆样本中的应用。使用纳米粒子跟踪分析（NanoSight）和 Western blot 来评估分离出的外泌体的效率和纯度。在我们的条件下，分离出的颗粒的大小分布合适（40-150nm），且 ExoQuickTM 外泌体沉淀溶液（EXQ）产生了相对较高产量的外泌体。然而，所有四种试剂盒都普遍存在白蛋白杂质，而总外泌体分离试剂盒（TEI）则产生了相对较纯的分离物。我们进一步进行了 Illumina 测序结合 RT-qPCR，以确定这些试剂盒进行 miRNA 分析的能力。虽然试剂盒之间的外泌体 miRNA 图谱和特定 miRNAs 具有显著相关性，但方法不同，相关性也不同。exoRNeasy 血清/血浆 Midi 试剂盒（EXR）和 EXQ 在外泌体特异性 miRNA 回收方面表现更好。特定 miRNA 测量的内标 CV 分别为 0.88-3.82%、1.19-3.77%、0-2.70%和 1.23-9.11%，用于 EXR、TEI、EXQ 和 RIBO™ 外泌体分离试剂（REI）。在每种试剂盒中，血清产生的外泌体和外泌体 miRNA 丰度均高于血浆，但白蛋白杂质更多。总之，我们的数据为循环外泌体 miRNA 的疾病生物标志物鉴定的方法学提供了一些有价值的指导。
