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Detection of single exosomes in microfluidic droplets by RT-PCR amplification of 18S RNA content
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-7510-0864
Karolinska Intitutet, Department of Oncology and Pathology.
Karolinska Intitutet, Department of Oncology and Pathology.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-5232-0805
(English)Manuscript (preprint) (Other academic)
Abstract [en]

We present a workflow for reverse transcription-PCR (RT-PCR) in microfluidic droplets to identify exosomes based on their RNA content. Available techniques for exosome detection have been limited to size or surface markers which limit their diagnostic capabilities. Exosome detection based on RNA content could be developed to be used as a diagnostic, prognostic or predictive tool for cancer based on specific RNA biomarkers in liquid biopsies. In this manuscript we demonstrate a high throughput method for the amplification of exosome derived 18S RNA in microfluidic droplets. Automated image analysis using open source software was applied to distinguish and count PCR-positive droplets with fluorescent intensity over a set threshold. We benchmark our workflow against picoliter scale RT-PCR on serially diluted exosome samples and demonstrate the ability of the droplet based workflow to correctly rank exosome samples based on exosome concentration.  This represents a key step towards a quantitative analysis of exosomal RNA content and the sorting of single exosomes by their RNA content.

Keyword [en]
Droplet Microfluidics, Exosomes, RT-PCR
National Category
Diagnostic Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-216586OAI: oai:DiVA.org:kth-216586DiVA: diva2:1151536
Note

QC 20171023

Available from: 2017-10-23 Created: 2017-10-23 Last updated: 2017-10-24Bibliographically approved
In thesis
1. Droplet Microfluidics reverse transcription and PCR towards Single Cell and Exosome Analysis
Open this publication in new window or tab >>Droplet Microfluidics reverse transcription and PCR towards Single Cell and Exosome Analysis
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Miniaturization of biological analysis is a trend in the field of biotechnology aiming to increase resolution and sensitivity in biological assays. Decreasing the reaction volumes to analyze fewer analytes in each reaction vessel enables the detection of rare analytes in a vast background of more common variants. Droplet microfluidics is a high throughput technology for the generation, manipulation and analysis of picoliter scale water droplets an in immiscible oil. The capacity for high throughput processing of discrete reaction vessels makes droplet microfluidics a valuable tool for miniaturization of biological analysis.

In the first paper, detection methods compatible with droplet microfluidics was expanded to include SiNR FET sensors. An integrated droplet microfluidics SiNR FET sensor device capable of extracting droplet contents, transferring a train of droplets to the SiNR to measure pH was implemented and tested. In paper II, a workflow was developed for scalable and target flexible multiplex droplet PCR using fluorescently color-coded beads for target detection. The workflow was verified for concurrent detection of two microorganisms infecting poultry. The detection panel was increased to multiple targets in one assay by the use of target specific capture probes on color-coded detection beads.   In paper III, droplet microfluidics has been successfully applied to single cell processing, demonstrated in paper III, where reverse transcription was performed on 65000 individually encapsulated mammalian cells. cDNA yield was approximately equivalent for reactions performed in droplets and in microliter scale. This workflow was further developed in paper IV to perform reverse transcription PCR in microfluidic droplets for detection of exosomes based on 18S RNA content. The identification of single exosomes based on RNA content can be further developed to detect specific RNA biomarkers for disease diagnostics.

Droplet microfluidics has great potential for increasing resolution in biological analysis and to become a standard tool in disease diagnostics and clinical research.

 

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. 69 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:15
Keyword
Droplet microfluidics, Reverse transcription, Droplet PCR, High Throughput biology, Single cell Analysis, Exosomes
National Category
Other Engineering and Technologies not elsewhere specified
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-216669 (URN)978-91-7729-577-8 (ISBN)
Public defence
2017-11-17, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20171024

Available from: 2017-10-24 Created: 2017-10-23 Last updated: 2017-10-26Bibliographically approved

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Jönsson, Håkan

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