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Optimization of small RNA library preparation protocol from human urinary exosomes
Dolores Olivares1, Javier Perez-Hernandez1,2, Daniel Perez-Gil1,3
1Cardiometabolic and Renal Risk Research Group, INCLIVA Biomedical Research Institute, Avd. Menéndez Pelayo, accesorio 4, 46010, Valencia, Spain.
Journal of Translational Medicine
|March 20, 2020
Summary
Optimizing small RNA (sRNA) library preparation for urinary exosomes enhances miRNA sequencing. Adding a gel purification step removes adapter-dimers and significantly increases miRNA reads for better biomarker discovery.
Area of Science:
- Biochemistry
- Molecular Biology
- Genomics
Background:
- Exosomes contain potential disease biomarkers, but low RNA yields hinder analysis.
- MicroRNA (miRNA) sequencing from exosomes is promising for biomarker discovery.
- Efficient small RNA (sRNA) library preparation is crucial for exosome-based miRNA analysis.
Purpose of the Study:
- To optimize a small RNA library preparation protocol for next-generation sequencing (NGS) of miRNA from urinary exosomes.
- To improve RNA analysis and quantification from low-yield exosome samples.
- To enhance the yield of miRNA reads for biomarker identification.
Main Methods:
- Utilized urinary exosome samples from 24 donors.
- Extracted RNA using column-based methods and assessed quality via spectrophotometry and Bioanalyzer.
- Prepared libraries using the CleanTag small RNA library protocol with modifications, including gel purification.
Main Results:
- Achieved high-quality sequencing libraries from urinary exosomes using the optimized protocol.
- Incorporating gel purification completely removed adapter-dimers from cDNA libraries.
- Observed a significant 37% increase in miRNA reads, augmenting overall small RNA mapped reads without altering the tagged miRNA population.
Conclusions:
- Developed an optimized sRNA library preparation workflow for exosome-associated miRNA analysis via NGS.
- The protocol yields a high number of miRNA mapped reads.
- This method is suitable for researchers seeking to improve miRNA biomarker discovery from urinary exosomes.
