Detection of single exosomes in microfluidic droplets by RT-PCR amplification of 18S RNA content

• 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.

Subject headings

TEKNIK OCH TEKNOLOGIER  -- Miljöbioteknik -- Diagnostisk bioteknologi (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Environmental Biotechnology -- Diagnostic Biotechnology (hsv//eng)

Keyword

Droplet Microfluidics

Exosomes

RT-PCR

Bioteknologi

Biotechnology

Publication and Content Type

vet (subject category)

ovr (subject category)