Institute for Molecular Medicine Finland FIMM, HiLIFE, University of Helsinki, 00290 Helsinki, Finland.
Institute for Molecular Medicine Finland FIMM, EV and HiPREP Core, University of Helsinki, 00290 Helsinki, Finland.
Genes (Basel). 2023 Jul 8;14(7):1415. doi: 10.3390/genes14071415.
Urinary extracellular vesicles (uEV) hold non-invasive RNA biomarkers for genitourinary tract diseases. However, missing knowledge about reference genes and effects of preanalytical choices hinder biomarker studies. We aimed to assess how preanalytical variables (urine storage temperature, isolation workflow) affect diabetic kidney disease (DKD)-linked miRNAs or kidney-linked miRNAs and mRNAs (kidney-RNAs) in uEV isolates and to discover stable reference mRNAs across diverse uEV datasets. We studied nine raw and normalized sequencing datasets including healthy controls and individuals with prostate cancer or type 1 diabetes with or without albuminuria. We focused on kidney-RNAs reviewing literature for DKD-linked miRNAs from kidney tissue, cell culture and uEV/urine experiments. RNAs were analyzed by expression heatmaps, hierarchical clustering and selecting stable mRNAs with normalized counts (>200) and minimal coefficient of variation. Kidney-RNAs were decreased after urine storage at -20 °C vs. -80 °C. Isolation workflows captured kidney-RNAs with different efficiencies. Ultracentrifugation captured DKD -linked miRNAs that separated healthy and diabetic macroalbuminuria groups. Eleven mRNAs were stably expressed across the datasets. Hence, pre-analytical choices had variable effects on kidney-RNAs-analyzing kidney-RNAs complemented global correlation, which could fade differences in some relevant RNAs. Replicating prior DKD-marker results and discovery of candidate reference mRNAs encourages further uEV biomarker studies.
尿细胞外囊泡（uEV）携带有泌尿生殖道疾病的非侵入性 RNA 生物标志物。然而，参考基因的缺失知识和分析前选择的影响阻碍了生物标志物的研究。我们旨在评估分析前变量（尿液储存温度、分离工作流程）如何影响糖尿病肾病（DKD）相关的 miRNA 或尿细胞外囊泡中与肾脏相关的 miRNA 和 mRNAs（肾脏-RNAs），并发现跨多种 uEV 数据集稳定的参考 mRNAs。我们研究了包括健康对照者和前列腺癌或 1 型糖尿病患者（有或无白蛋白尿）在内的 9 个原始和标准化测序数据集。我们重点研究了肾脏-RNAs，回顾了肾脏组织、细胞培养和 uEV/尿液实验中与 DKD 相关的 miRNA 的文献。通过表达热图、层次聚类和选择具有标准化计数（>200）和最小变异系数的稳定 mRNAs 来分析 RNA。与 -80°C 相比，-20°C 储存尿液后肾脏-RNAs 减少。分离工作流程以不同的效率捕获肾脏-RNAs。超速离心分离了健康和糖尿病大量白蛋白尿组的 DKD 相关 miRNA。11 个 mRNAs 在所有数据集上稳定表达。因此，分析前的选择对肾脏-RNAs 有不同的影响，分析肾脏-RNAs 补充了全局相关性，这可能会淡化一些相关 RNA 的差异。复制先前 DKD 标志物的结果和发现候选参考 mRNAs 鼓励进一步进行 uEV 生物标志物研究。
