Abstract
Development of a robust automated platform for enrichment of extracellular vesicles from low sample volume that matches the needs for next-generation sequencing could remove major hurdles for genomic biomarker discovery. Here, we document a protocol for urinary EVs enrichment by utilizing an automated microfluidic system, termed acoustic trap, followed by next-generation sequencing of microRNAs (miRNAs) for biomarker discovery. Specifically, we compared the sequencing output from two small RNA library preparations, NEXTFlex and CATS, using only 130 pg of input total RNA. The samples prepared using NEXTflex was found to contain larger number of unique miRNAs that was the predominant RNA species whereas rRNA was the dominant RNA species in CATS prepared samples. A strong correlation was found between the miRNA expressions of the acoustic trap technical replicate in the NEXTFlex prepared samples, as well as between the acoustic trap and ultracentrifugation enrichment methods. Together, these results demonstrate a robust and automated strategy for biomarker discovery from small volumes of urine.
Highlights
We estimated the total RNA isolated from the acoustic trapped sample by multiplying the average relative abundance of miR-16/21/24 (0.57%±0.15%) to the 138 ng of total RNA obtained from ultracentrifugation sample (Fig 2b) which resulted in 0.79 ng of total RNA
This study demonstrated that by utilizing the NEXTflex library preparation kit as a downstream pipeline after automated acoustic trapping, small RNA libraries can be successfully constructed from as little as 130 pg of total RNA, equivalent to 1.7 mL of urine
Though the library preparation may benefit from additional size-selection steps to remove adapter dimers, we obtained a sufficient number of reads for acoustic trapped samples
Summary
A single spot urine sample collected from a heathy male donor with written consent and approval by Regional Ethical Review Board in Lund # 2013–400 was immediately centrifuged at 2,000xg for ten minutes at room temperature to remove cell debris while preserving the small and large EVs in the sample. Nanoparticle-tracking analysis (NTA) was performed using NanoSight LM10 (Malvern, UK) on urine samples diluted eight-times (0.125x of stock) with PBS using continuous flush mode and measured five times.
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