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PLoS One. 2019 May 29;14(5):e0217507. doi: 10.1371/journal.pone.0217507. eCollection 2019.

A urinary extracellular vesicle microRNA biomarker discovery pipeline; from automated extracellular vesicle enrichment by acoustic trapping to microRNA sequencing.

Author information

1
Department of Translational Medicine, Lund University, Malmö, Sweden.
2
Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden.
3
Department of Biomedical Engineering, Lund University, Lund, Sweden.
4
Urology Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
5
Genitourinary Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
6
Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
7
Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom.
8
Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden.

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.

Conflict of interest statement

Thomas Laurell and Hans Lilja are founders and own stock in AcouSort AB and Mikael Evander owns stock in AcouSort AB. AcouSort AB is a spin-off company from Lund University that manufactures and markets instrumentation for acoustic trapping. Unrelated to the current study, Hans Lilja holds patents on assays for intact PSA (“Antibody, immunoassay and method for prostate cancer detection.” U.S. Patent No. US 7,872,104 B2) and a patent for a statistical method to detect prostate cancer (Methods and Apparatuses For Predicting Risk Of Prostate Cancer And Prostate Gland Volume. U.S. Patent No. US. 9,672,329 B2) commercialized by OPKO Health. Hans Lilja receives royalties from sales of the test and has stock in OPKO Health. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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