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 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 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. Here, we further studied urine-derived exosomes and their mRNA content as a highly promising dia
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 via 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.
Core Tip: Recently, it was demonstrated that analysis of urine-derived exosomal mRNA allows early detection of kidney allograft rejection. We further studied exosomes and their mRNA content as a highly promising diagnostic modality. This included stability studies of urine samples and exo-mRNA upon transportation from the point of collection to centralized testing facility, short-term storage of urine at different conditions upon receipt till the point molecular assay is performed, and effects of various interfering substances on the quantitative polymerase chain reaction assay. mRNA from the urine-derived exosomes was proven to be stable across broad range of conditions and produce robust results in post-transplant monitoring assays.