McFaul Matt, Ventura Chris, Evans Sean, Dundar Halil, Rumpler Marc J, McCloskey Christopher, Lowe Dave, Vlassov Alexandre V
Department of Research and Development, Thermo Fisher Scientific, West Hills, CA 91304, United States.
World J Methodol. 2023 Dec 20;13(5):492-501. doi: 10.5662/wjm.v13.i5.492.
Exosomes are 30-150 nm nanovesicles with sophisticated nucleic acids cargo, actively secreted by all cells within human body, and found in abundance in all body fluids, including urine. These extracellular vesicles have tremendous potential for next generation diagnostics, theoretically enabling noninvasive assessment of organ and tissue function liquid biopsy analysis.
Recently, feasibility of an exosomal molecular test was demonstrated for post-organ transplant monitoring: Analysis of urine-derived exosomal mRNA cargo allowed early detection of kidney allograft rejection. Here, we further studied urine-derived exosomes and their mRNA content as a highly promising diagnostic modality. This included stability studies of urine samples and exosomal mRNA upon transportation from the point of collection to a centralized testing facility, short-term storage of urine at different conditions upon receipt till the point molecular assay is performed, and effects of various potentially interfering substances on the downstream quantitative polymerase chain reaction (qPCR) assay.
The urine specimens were stored at various conditions and pre-processed in different ways. Next, samples were passed through the columns to capture all extracellular vesicles, the vesicles were lysed to release their content and the exosomal RNA was purified on the mini-columns, reverse transcription was performed, next pre-amplification, followed by a qPCR analysis for a panel of mRNA markers.
To ensure exosomal RNA integrity, the harvested urine specimens should be shipped refrigerated, by overnight delivery. Urine can next be stored at the test site for up to 1 wk at 4 °C, and long term should be frozen at -80 °C. Urine specimens must be centrifuge at low G-force to deplete cells and debris, to ensure consistent top results in downstream molecular assays. All commonly used medications (tacrolimus, cyclosporin A, mycophenolic acid, everolimus, sirolimus, ascomycin, teriflunomide) were tested and confirmed that they do not cause assay interference.
mRNA from urine-derived exosomes was shown to be stable across a broad range of conditions and produced accurate results when analyzed qPCR assay for detection of kidney allograft rejection. We identified the most optimal conditions for every step of the process, ensuring pre-analytical sample integrity and robust qPCR results.
外泌体是直径为30 - 150纳米的纳米囊泡，携带着复杂的核酸物质，由人体内所有细胞主动分泌，在包括尿液在内的所有体液中大量存在。这些细胞外囊泡在下一代诊断中具有巨大潜力，理论上能够实现对器官和组织功能的无创评估及液体活检分析。
最近，已证明外泌体分子检测用于器官移植后监测的可行性：分析尿液来源的外泌体mRNA成分可早期检测肾移植排斥反应。在此，我们进一步研究尿液来源的外泌体及其mRNA含量作为一种极有前景的诊断方式。这包括尿液样本和外泌体mRNA在从采集点运输到集中检测设施过程中的稳定性研究、收到尿液后在不同条件下短期储存直至进行分子检测以及各种潜在干扰物质对下游定量聚合酶链反应（qPCR）检测的影响。
尿液标本在各种条件下储存并采用不同方式进行预处理。接下来，样本通过柱子以捕获所有细胞外囊泡，将囊泡裂解以释放其内容物，在外泌体RNA在微型柱上进行纯化，进行逆转录，然后进行预扩增，随后对一组mRNA标志物进行qPCR分析。
为确保外泌体RNA的完整性，采集的尿液标本应冷藏运输，隔夜送达。尿液接下来可在检测地点4℃下储存长达1周，长期应在 - 80℃下冷冻。尿液标本必须以低离心力离心以去除细胞和碎片，以确保下游分子检测获得一致的最佳结果。对所有常用药物（他克莫司、环孢素A、霉酚酸、依维莫司、西罗莫司、子囊霉素、特立氟胺）进行了检测，证实它们不会引起检测干扰。
尿液来源外泌体的mRNA在广泛条件下显示出稳定性，在用于检测肾移植排斥反应的qPCR分析中产生准确结果。我们确定了该过程每个步骤的最佳条件，确保分析前样本的完整性和可靠的qPCR结果。
