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Micromachines (Basel). 2019 Mar 27;10(4). pii: E215. doi: 10.3390/mi10040215.

Multiplexed PCR-Free Detection of MicroRNAs in Single Cancer Cells Using a DNA-Barcoded Microtrough Array Chip.

Wang N1, Lu Y2, Chen Z3, Fan R4,5,6,7.

Author information

1
Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. wangnayi@gmail.com.
2
Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. luyao@dicp.ac.cn.
3
Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. zhuo.chen@yale.edu.
4
Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA. rong.fan@yale.edu.
5
Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA. rong.fan@yale.edu.
6
Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT 06520, USA. rong.fan@yale.edu.
7
Human and Translational Immunology Program, Yale University School of Medicine, New Haven, CT 06520, USA. rong.fan@yale.edu.

Abstract

MicroRNAs are a class of small RNA molecules that regulate the expression of mRNAs in a wide range of biological processes and are implicated in human health and disease such as cancers. How to measure microRNA profiles in single cells with high throughput is essential to the development of cell-based assays for interrogating microRNA-mediated intratumor heterogeneity and the design of new lab tests for diagnosis and monitoring of cancers. Here, we report on an in situ hybridization barcoding workflow implemented in a sub-nanoliter microtrough array chip for high-throughput and multiplexed microRNA detection at the single cell level. The microtroughs are used to encapsulate single cells that are fixed, permeabilized, and pre-incubated with microRNA detection probes, each of which consists of a capture strand complementary to specific microRNA and a unique reporter strand that can be photocleaved in the microtroughs and subsequently detected by an array of DNA barcodes patterned on the bottom of the microtroughs. In this way, the measurement of reporter strands released from single cells is a surrogate for detecting single-cell microRNA profiles. This approach permits direct measurement of microRNAs without PCR amplification owing to the small volume (<1 nL) of microtroughs. It offers high throughput and high multiplexing capability for evaluating microRNA heterogeneity in single cells, representing a new approach toward microRNA-based diagnosis and monitoring of complex human diseases.

KEYWORDS:

DNA barcoding; microRNA; microtrough arrays; single-cell analysis

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