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Issue 24, 2018
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Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip

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Abstract

Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it remains a daunting challenge to quantitatively measure molecular contents of EVs including tumor-associated mRNAs. Herein, we report a configurable microwell-patterned microfluidic digital analysis platform combined with a dual-probe hybridization assay for PCR-free, single-molecule detection of specific mRNAs in EVs. The microwell array in our device is configurable between the flow-through assay mode for enhanced hybridization capture and tagging of mRNAs and the digital detection mode based on femtoliter-scale enzymatic signal amplification for single-molecule counting of surface-bound targets. Furthermore, a dual-probe hybridization assay has been developed to enhance the sensitivity of the digital single-molecule detection of EV mRNAs. Combining the merits of the chip design and the dual-probe digital mRNA hybridization assay, the integrated microfluidic system has been demonstrated to afford quantitative detection of synthetic GAPDH mRNA with a LOD as low as 20 aM. Using this technology, we quantified the level of GAPDH and EWS-FLI1 mRNAs in EVs derived from two cell lines of peripheral primitive neuroectodermal tumor (PNET), CHLA-9 and CHLA-258. Our measurements detected 64.6 and 43.5 copies of GAPDH mRNA and 6.5 and 0.277 copies of EWS-FLI1 fusion transcripts per 105 EVs derived from CHLA-9 and CHLA-258 cells, respectively. To our knowledge, this is the first demonstration of quantitative measurement of EWS-FLI1 mRNA copy numbers in Ewing Sarcoma (EWS)-derived EVs. These results highlight the ultralow frequency of tumor-specific mRNA markers in EVs and the necessity of developing highly sensitive methods for analysis of EV mRNAs. The microfluidic digital mRNA analysis platform presented here would provide a useful tool to facilitate quantitative analysis of tumor-associated EV mRNAs for liquid biopsy-based cancer diagnosis and monitoring.

Graphical abstract: Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip

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Supplementary files

Article information


Submitted
09 Oct 2018
Accepted
19 Nov 2018
First published
20 Nov 2018

Lab Chip, 2018,18, 3790-3801
Article type
Paper

Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip

P. Zhang, J. Crow, D. Lella, X. Zhou, G. Samuel, A. K. Godwin and Y. Zeng, Lab Chip, 2018, 18, 3790
DOI: 10.1039/C8LC01071D

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