A path to ultra-low input microRNA sequencing from urinary extracellular vesicles after acoustic trap enrichment

Background: There are increasing recognition that microRNA (miRNA) contained in extracellular vesicles (EVs) play a pivotal role in disease progression. The challenge to use miRNA in EVs as a biomarker has been hampered by a lack of a robust method to enrich and sequence miRNA from minute quantities...

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Bibliographic Details
Published in:Journal of extracellular vesicles 2018-01, Vol.7, p.220-220
Main Authors: Ku, Anson T, Evander, Mikael, Persson, Margareta, Lilja, Hans, Laurell, Thomas, Ceder, Yvonne
Format: Article
Language:English
Subjects:
Acoustics
Extracellular vesicles
Microfluidics
MicroRNAs
miRNA
Nucleotide sequence
Polymerase chain reaction
Ultracentrifugation
Urine
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Summary:Background: There are increasing recognition that microRNA (miRNA) contained in extracellular vesicles (EVs) play a pivotal role in disease progression. The challenge to use miRNA in EVs as a biomarker has been hampered by a lack of a robust method to enrich and sequence miRNA from minute quantities of initial samples. Utilizing the acoustic trap, which is a novel microfluidic technology that utilizes ultrasonic waves to enrich extracellular vesicles, we enriched urinary EVs in a contact-free and automated manner. Next, we compared the performance of two different small RNA library preparations using 130 pg of input RNA derived from urinary EVs. In addition, we compared the miRNA obtained from acoustic trap to ultracentrifugation to determine the performance of the acoustic trap method. Methods: Urinary extracellular vesicles were enriched from approximately 2.5 mL of urine by acoustic trap and ultracentrifugation follow by RNase A treatment. Total RNA was extracted using Single Cell RNA extraction kit (Norgen) and approximately 130 pg of RNA was used for library construction using the small RNA library preparation kits, NEXTFlex (Perkin Elmers) and CATs (Diagenode). Specifically, two library replicates were constructed from acoustic trapped sample and one from the ultracentrifugation enriched sample. The library profiles were confirmed by Bioanalyzer and Qubit DNA assay and sequenced on an Illumina NextSeq platform. The miRNA expression of three miRNAs, has-miR-16, 21, and 24, was validated using qRT-PCR. Results: Small RNA libraries were successfully constructed from 130 pg of RNA derived from acoustic trap and ultracentrifugation method using both NEXTFlex and CATS small RNA library preparation kits. Three different miRNAs were used to validate the finding by qRT-PCR. Summary/Conclusion: Acoustic trap enrichment of urinary EVs can produce sufficient quantities of RNA for miRNA sequencing using either NEXTFlex or CATS small RNA library preparation.
ISSN:2001-3078