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Impact of Extracellular Vesicle Isolation Methods on Downstream Mirna Analysis in Semen: A Comparative Study
Marina Mercadal1, Carolina Herrero2, Olga López-Rodrigo3
1Human Molecular Genetics Group-Bellvitge Biomedical Research Institute (IDIBELL), 08908 Hospitalet de Llobregat, Barcelona, Spain.
Abstract:
Seminal plasma (SP) contains a unique concentration of miRNA, mostly contained in small extracellular vesicles (sEVs) such as exosomes, some of which could be clinically useful for diagnosis and/or prognosis of urogenital diseases such as prostate cancer (PCa). We optimized several exosome-EV isolation technologies for their use in semen, evaluating EV purifying effectiveness and impact on the downstream analysis of miRNAs against results from the standard ultracentrifugation (UC) method to implement the use of SP sEV_miRNAs as noninvasive biomarkers for PCa. Our results evidenced that commercial kits designed to isolate exosomes/EVs from blood or urine are mostly applicable to SP, but showed quantitative and qualitative variability between them. ExoGAG 3500× g and the miRCURY Cell/Urine/CSF 1500× g methods resulted as equivalent alternative procedures to UC for isolating exosomes/sEVs from semen for nanoparticle characteristics and quality of RNA contained in vesicles. Additionally, the expression profile of the altered semen sEV-miRNAs in PCa varies depending on the EV isolation method applied. This is possibly due to different extraction techniques yielding different proportions of sEV subtypes. This is evidence that the exosome-EV isolation method has a significant impact on the analysis of the miRNAs contained within, with important consequences for their use as clinical biomarkers. Therefore, miRNA analysis results for EVs cannot be directly extrapolated between different EV isolation methods until clear markers for delineation between microvesicles and exosomes are established. However, EV extraction methodology affects combined models (semen exosome miRNA signatures plus blood Prostate specific antigen (PSA) concentration for PCa diagnosis) less; specifically our previously described (miR-142-3p + miR-142-5p + miR-223-3p + PSA) model functions as molecular marker from EVs from any of the three isolation methods, potentially improving the efficiency of PSA PCa diagnosis.
Insights
Optimizing seminal plasma exosome isolation methods is crucial for accurate prostate cancer (PCa) biomarker analysis. Certain methods show promise as noninvasive diagnostic tools, improving upon traditional prostate-specific antigen (PSA) testing.
Area of Science:
- Biochemistry
- Molecular Biology
- Urology
Background:
- Seminal plasma (SP) contains microRNAs (miRNAs) within small extracellular vesicles (sEVs), offering potential as noninvasive biomarkers for urogenital diseases like prostate cancer (PCa).
- Standard ultracentrifugation (UC) is a common method for isolating sEVs, but its efficiency and impact on downstream miRNA analysis require comparison with newer techniques.
Purpose of the Study:
- To optimize and evaluate exosome-EV isolation technologies for semen, assessing their effectiveness and impact on miRNA analysis for PCa biomarker development.
- To compare the performance of commercial isolation kits and specific methods (ExoGAG, miRCURY) against the standard UC method.
Main Methods:
- Evaluation of commercial exosome-EV isolation kits and specific methods (ExoGAG 3500×g, miRCURY 1500×g) using semen.
- Comparison of isolated EV characteristics and RNA quality against the ultracentrifugation (UC) standard.
- Analysis of miRNA expression profiles from isolated EVs and assessment of their impact on a combined PCa diagnostic model.
Main Results:
- Commercial kits are applicable to SP but exhibit variability; ExoGAG and miRCURY methods are viable alternatives to UC for EV isolation from semen.
- The choice of EV isolation method significantly impacts the detected semen sEV-miRNA expression profiles in PCa.
- A combined biomarker model (semen exosome miRNAs + blood Prostate-Specific Antigen (PSA)) demonstrated robustness across different EV isolation methods, potentially enhancing PCa diagnosis.
Conclusions:
- EV isolation methodology critically influences miRNA analysis results, necessitating careful consideration for clinical biomarker applications.
- Standardization of EV isolation techniques or development of markers to distinguish EV subtypes is needed for reliable miRNA biomarker extrapolation.
- The tested EV isolation methods minimally affect a combined miRNA-PSA biomarker model, suggesting its potential for improved noninvasive PCa diagnosis.
