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Urine exosome mRNA-based test for monitoring kidney allograft rejection:Effects of sample transportation and storage,and interference substances

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World Journal of Methodology 2023年第5期13卷 492-501页

作者： Matt McFaul Chris Ventura Sean Evans Halil Dundar Marc J Rumpler Christopher McCloskey dave lowe Alexandre V Vlassov Department of Research and Development Thermo Fisher ScientificWest HillsCA 91304United States

BACKGROUND 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 *** extracellular vesicles have tremendous potential for next generation diagnostics,theoretically enabling noninvasive assessment of organ and tissue function via liquid biopsy *** Recently,feasibility of an exosomal molecular test was demonstrated for postorgan transplant monitoring:Analysis of urine-derived exosomal mRNA cargo allowed early detection of kidney allograft ***,we further studied urine-derived exosomes and their mRNA content as a highly promising diagnostic *** 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)*** The urine specimens were stored at various conditions and pre-processed in different ***,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 RESULTS To ensure exosomal RNA integrity,the harvested urine specimens should be shipped refrigerated,by overnight *** can next be stored at the test site for up to 1 wk at 4°C,and long term should be frozen at-80°*** specimens must be centrifuge at low G-force to deplete cells and debris,to ensure consistent top results in downstream molecular *** commonly used medications(tacrolimus,cyclosporin A,mycophenolic acid,everolimus,sirolimus,ascomycin,teriflunomide)were tested and confirmed that they do n

关键词： Kidney allograft Post-transplant monitoring Liquid biopsy Exosome mRNA Quantitative polymerase chain reaction

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