Comparative analysis of EV isolation procedures for miRNAs detection in serum samples

Area of Science:

• Biochemistry
• Molecular Biology
• Biomarker Discovery

Background:

• Extracellular vesicles (EVs) are promising non-invasive biomarkers.
• Current EV isolation methods are often time-consuming and not clinically practical.
• Efficient enrichment of EVs from serum is crucial for circulating microRNA (miRNA) analysis.

Purpose of the Study:

• To identify a quick, easy, and economical method for enriching extracellular vesicles (EVs) from frozen human serum for microRNA (miRNA) analysis.
• To compare the efficiency of various EV isolation protocols and commercial kits against ultracentrifugation.
• To validate the utility of the optimized method for miRNA biomarker assessment in clinical samples.

Main Methods:

• Compared precipitation, column, and filter-based EV isolation methods with ultracentrifugation.
• Assessed EV purity and quantity using nanoparticle tracking analysis, western blot, and cytometry.
• Quantified specific miRNAs in isolated EV fractions and total serum using qPCR.
• Evaluated miRNA stability in long-term stored serum samples using the selected isolation method.

Main Results:

• Precipitation-based methods, particularly using polyethylene glycol (PEG), effectively enriched EVs for specific encapsulated miRNAs (miR-126, miR-30c, miR-143).
• PEG precipitation demonstrated comparable or superior protein and miRNA yield to other commercial reagents at a significantly lower cost.
• Isolated EVs allowed for miRNA assessment in serum samples stored for up to 8 years.
• A method was developed to estimate miRNA encapsulation degree based on EV detection fold-change versus serum.

Conclusions:

• Polyethylene glycol (PEG) precipitation is a suitable, cost-effective, and efficient method for enriching serum extracellular vesicles (EVs) for microRNA (miRNA) analysis.
• The developed method is robust, enabling reliable miRNA biomarker assessment from long-term stored frozen serum samples.
• This approach facilitates the clinical translation of EV-based miRNA diagnostics.