Published online Dec 20, 2023. doi: 10.5662/wjm.v13.i5.492
Peer-review started: July 21, 2023
First decision: August 31, 2023
Revised: September 7, 2023
Accepted: October 23, 2023
Article in press: October 23, 2023
Published online: December 20, 2023
Exosomes are nano-sized extracellular vesicles with nucleic acid and protein 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 via 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. Taking into account the novelty of this approach, urine and in particular extracellular vesicles with their diverse RNA cargo have to be better characterized to ensure robustness of this molecular assay.
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 temperatures and 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 centrifuged 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 the urine-derived exosomes was proven to be stable across a broad range of conditions and produce robust results in molecular post-transplant monitoring assays. We identified optimal conditions for every step of the workflow, ensuring pre-analytical sample integrity and robust downstream qPCR results.
Exosomes and in particular their mRNA cargo have the potential to revolutionize post-transplant monitoring, and detect early rejection events for kidney as well as other allografts- based on molecular analysis of urine, saliva and other body fluids.