Date Published: May 29, 2019
Publisher: Public Library of Science
Author(s): Anson Ku, Naveen Ravi, Minjun Yang, Mikael Evander, Thomas Laurell, Hans Lilja, Yvonne Ceder, Bernard Mari.
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.
Extracellular vesicles (EVs) are secreted cellular products that harbor proteins, lipids, DNA and RNA, especially small RNA such as microRNA (miRNA) within the phospholipid bilayer that protects them from the degradative effect of circulating proteases and nucleases . Recent research has shown that miRNAs in EVs play a pivotal role in the progression of various diseases. In cancer, intravesicular miRNAs have been shown to confer drug resistance to surrounding tumor cell population, induce local vascular expansion, trigger immune-shielding and prime distant metastatic niches . As a result of its systemic availability and biological role, EV miRNAs could serve as a valuable source of biomarkers and provide prognostic information.
Overcoming current limitations to enrich EVs is critical for translating EV-based diagnostics into clinical practice. We previously showed that EVs, ranging widely in size from exosomes to microvesicles, can be enriched using the acoustic trap method . However, prerequisites for biomarker discovery and clinical applications, such as detailed protocols incorporating an efficient and robust EV enrichment method combined with sensitive downstream sequencing pipeline are lacking. In addition, isolation of EVs from urine is particularly challenging due to the presence of Tamm-Horsfall protein that can sequester EVs and the large concentration range that EVs can present in urine samples . Here we demonstrate that urinary EVs can be enriched using acoustic trapping technology and subsequently analyzed by next generation sequencing using an estimated 130–150 pg of total RNA based on Qubit and qPCR or Bioanalyzer alone.
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. Thus, acoustic trap, in conjunction with NEXTFlex can be used for high throughput and automated miRNA biomarker discovery or clinical workflow in the future.