(W1030-03-19) Comparison of Exosome Isolation Methods for the Analysis of a Disease Biomarker in Serum and Urine by PCR Methodologies

Purpose: Extracellular vehicles (EVs) are small lipid enclosed carriers of bioactive proteins, lipids and nucleic acids that play an important role in the communication between cells, organs and organisms (1). Among EVs, exosomes have recently emerged as promising biomarkers for diseases. To this end, knowledge regarding their isolation, characterization and content could offer opportunities for disease detection and pharmacodynamic assessment of different therapies (2).

Methods: There are currently multiple protocols and kits available for exosome isolation and mRNA/miRNA content extraction. However, the performance of these methods to ultimately extract sufficient material for analysis of a large number of clinical samples remains unwell characterized. In this study, five exosome isolation protocols were compared, along with four mRNA extraction methods using serum and urine human samples (Fig 1). The extracted material was further analyzed by RT-qPCR and RT-ddPCR to quantify 2 target genes of interest relative to a housekeeping gene.

Results: Gene expression analysis by RT-qPCR shows that exosome and RNA isolated with the exoRNeasy kit from Qiagen resulted in higher quantification of gene X, gene Y and gene Z in serum when compared to the other exosome isolation techniques. However, in urine, only genes X and Z were detectable using exoRneasy kit (Fig 2 and Fig 3). RT-ddPCR analysis show comparable results to the RT-qPCR.

Conclusion: Using optimized protocols for both the exosome and mRNA extraction, it was possible to obtain sufficient material, with an acceptable quality, to perform gene expression analysis for downstream pharmacodynamic assessment of a novel siRNA target molecule.

References: 1- Xia Li; et al. Challenges and opportunities in exosome research—Perspectives from biology, engineering, and cancer therapy. APL Bioeng 2019 Mar 27;3(1):011503.
2- Chen YF. et al. Exosomes: a review of biologic function, diagnostic and targeted therapy applications, and clinical trials. J Biomed Sci 31, 67 (2024).

ExosomesRNA and PCR Protocols used

RTqPCR analysis of Urine Exosomal RNA

RTqPCR analysis of Serum Exosomal RNA