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Updated: Dec 9, 2025
Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile
Published on: June 13, 2016
Comparison of serum exosome isolation methods on co-precipitated free microRNAs
Yirui Cheng1, Xiangyun Qu2, Zhaonan Dong2
1School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China.
Background:
Exosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown.
Methods:
In this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods.
Results:
Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37 °C for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used. In conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.
Insights
The ExoQuick kit co-isolates some free microRNAs (miRNAs) from serum, unlike ultracentrifugation (UC), which isolates very few. Incubation and RNase A treatment can reduce free miRNAs during exosome isolation.
Area of Science:
- Extracellular vesicle research
- Biomarker discovery
- Molecular biology
Background:
- Exosomes are nano-sized vesicles involved in intercellular communication via biomolecules like microRNAs (miRNAs).
- Exosomal miRNAs are crucial in disease development and hold potential as diagnostic biomarkers.
- Common exosome isolation methods include ultracentrifugation (UC) and precipitation kits (e.g., ExoQuick), but their efficiency in isolating free miRNAs is unclear.
Purpose of the Study:
- To compare the efficiency of UC and ExoQuick methods in co-isolating free miRNAs from human serum.
- To investigate factors affecting free miRNA co-precipitation during exosome isolation.
Main Methods:
- Exosomes were isolated from human serum spiked with cel-miR-39 using UC and ExoQuick.
- The proportion of co-precipitated cel-miR-39 (free miRNA) was quantified for each method.
- Effects of incubation at 37°C and RNase A treatment on free miRNA levels were assessed.
Main Results:
- ExoQuick co-isolated a small proportion of free miRNAs, whereas UC isolated negligible amounts.
- Incubation at 37°C for 1 hour reduced the proportion of free miRNAs.
- RNase A treatment decreased levels of exosomal miRNAs (miR-126, miR-152).
Conclusions:
- The choice of exosome isolation method significantly impacts the co-precipitation of free miRNAs.
- UC is more suitable for isolating exosomes with minimal free miRNA contamination compared to ExoQuick.
- Optimizing isolation conditions, such as temperature and enzyme treatment, may help reduce free miRNA contamination.
