Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease.
Comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease.
复制标题
尿细胞外囊泡分离方法在糖尿病肾病转录组生物学标志物研究中的比较。
DOI：
10.1002/jev2.12038
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发表时间：
2020-12
影响因子：
16
通讯作者：
Holthofer H
中科院分区：
文献类型：
--
作者：
Barreiro K;Dwivedi OP;Leparc G;Rolser M;Delic D;Forsblom C;Groop PH;Groop L;Huber TB;Puhka M;Holthofer H
尿细胞外囊泡（uEV）已成为肾损伤生物标志物的来源，有可能取代传统的侵入性技术，包括肾活检。然而，全面的研究表征uEV分离方法与患者样本是罕见的。在这里，我们比较了三种已建立的uEV分离工作流程的性能，以便随后用于转录组学分析，以发现糖尿病肾病中的生物标志物。我们收集了1型糖尿病伴大量白蛋白尿患者和健康对照者的尿样。我们通过静水滤过透析（HFD）、超离心（UC）和基于商业试剂盒的分离方法（NG）分离uEV，每种方法都有不同的尿液清除步骤。通过电子显微镜、纳米颗粒跟踪分析和蛋白质印迹分析纯化的EV。对分离的RNA进行miRNA和RNA测序。根据mRNA测序数据，HFD和UC样本显示出密切的相似性。与HFD或UC相比，NG样品具有较低的读取数和不同的mRNA含量。对于miRNA测序数据，所有方法都获得了令人满意的miRNA计数，但miRNA含量略有不同。这表明分离工作流程富集了富含miRNA的uEV制备组分的特定亚群。我们的数据表明，HFD、UC和基于试剂盒的方法是分离用于miRNA-seq的uEV的合适方法。然而，在我们的环境中，只有HFD和UC适合于mRNA-seq。
Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit‐ based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA‐rich uEV preparation components. Our data shows that HFD,UC and the kit‐based method are suitable methods to isolate uEV for miRNA‐seq. However, only HFD and UC were suitable for mRNA‐seq in our settings.
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影响因子：
16
作者：
Lötvall J;Hill AF;Hochberg F;Buzás EI;Di Vizio D;Gardiner C;Gho YS;Kurochkin IV;Mathivanan S;Quesenberry P;Sahoo S;Tahara H;Wauben MH;Witwer KW;Théry C
通讯作者：
Théry C
影响因子：
6
作者：
Ghai V;Wu X;Bheda-Malge A;Argyropoulos CP;Bernardo JF;Orchard T;Galas D;Wang K
通讯作者：
Wang K
影响因子：
16
作者：
Bachurski, Daniel;Schuldner, Maximiliane;von Strandmann, Elke Pogge
通讯作者：
von Strandmann, Elke Pogge
影响因子：
2.5
作者：
McNicholas, Kym;Li, Jordan Y. Z.;Gleadle, Jonathan M.
通讯作者：
Gleadle, Jonathan M.
影响因子：
16
作者：
Barreiro K;Dwivedi OP;Leparc G;Rolser M;Delic D;Forsblom C;Groop PH;Groop L;Huber TB;Puhka M;Holthofer H
通讯作者：
Holthofer H