A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents.
A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents.
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使用差分超速离心和三种商业试剂对血清外泌体分离的比较研究。
DOI：
10.1371/journal.pone.0170628
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Liu Y
中科院分区：
文献类型：
--
作者：
Helwa I;Cai J;Drewry MD;Zimmerman A;Dinkins MB;Khaled ML;Seremwe M;Dismuke WM;Bieberich E;Stamer WD;Hamrick MW;Liu Y
外泌体在细胞间信号传导中发挥作用，并作为可能的生物标志物。分离具有可靠质量和实质浓度的外泌体是一个重大挑战。我们的目的是比较三种不同的外泌体分离试剂盒提取的外泌体使用六种不同体积的非癌性人血清，使用miRCURY、ExoQuick和Invitrogen总外泌体分离试剂（Total Exosome Isolation Reagent）和差异超离心（UC），（5 ml、1 ml、500 μl、250 μl、100 μl和50 μl）和三种不同体积（1 ml、500 μl和100 μl）的6份从人供体采集的单独市售血清样品。较小的起始体积（100 μl和50 μl）用于模拟异质生物样品有限可用性的条件。基于大小、数量、ζ电位、CD 63和CD 9蛋白表达以及外泌体RNA（exRNA）质量和数量，使用几种互补方法表征分离的外泌体：使用ZetaView的纳米颗粒跟踪分析（NTA）、蛋白质印迹、透射电子显微镜（TEM）、Agilent Bioanalyzer系统和液滴数字PCR（ddPCR）。我们的NTA结果显示，所有分离技术均产生在预期尺寸范围（40-150 nm）内的外泌体。然而，与UC相比，对于所有血清体积，除了5 mL，三种试剂盒产生了显著更高的外泌体产量（80-300倍）。我们还发现，通过不同技术和血清体积分离的外泌体具有与先前研究相似的zeta电位。Western印迹分析和TEM免疫金标记证实了两种常见的外泌体蛋白标记物，CD 63和CD 9，在通过所有技术分离的样品中的表达。所有外泌体分离产生高质量的exRNA，其主要含有大小在25至200个核苷酸之间的峰值的小RNA。ddPCR结果表明，从相似的血清体积但不同的分离技术分离的外泌体提供了相似浓度的两种选择的exRNA：hsa-miR-16和hsa-miR-451。总之，三种商业外泌体分离试剂盒是UC的可行替代品，即使在有限量的生物样品可用时。
Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40–150 nm). The three kits, though, produced a significantly higher yield (80–300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.