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Detection of single exosomes in microfluidic droplets by RT-PCR amplification of 18S RNA content
KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, 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, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.ORCID-id: 0000-0001-5232-0805
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
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.

Emneord [en]
Droplet Microfluidics, Exosomes, RT-PCR
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-216586OAI: oai:DiVA.org:kth-216586DiVA, id: diva2:1151536
Merknad

QC 20171023

Tilgjengelig fra: 2017-10-23 Laget: 2017-10-23 Sist oppdatert: 2022-06-26bibliografisk kontrollert
Inngår i avhandling
1. Droplet Microfluidics reverse transcription and PCR towards Single Cell and Exosome Analysis
Åpne denne publikasjonen i ny fane eller vindu >>Droplet Microfluidics reverse transcription and PCR towards Single Cell and Exosome Analysis
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

 

 

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2017. s. 69
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:15
Emneord
Droplet microfluidics, Reverse transcription, Droplet PCR, High Throughput biology, Single cell Analysis, Exosomes
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-216669 (URN)978-91-7729-577-8 (ISBN)
Disputas
2017-11-17, Air & Fire, Tomtebodavägen 23A, Solna, 10:00 (engelsk)
Opponent
Veileder
Merknad

QC 20171024

Tilgjengelig fra: 2017-10-24 Laget: 2017-10-23 Sist oppdatert: 2022-06-26bibliografisk kontrollert

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