Multiplexed PCR-Free Detection of MicroRNAs in Single Cancer Cells Using a DNA-Barcoded Microtrough Array Chip
Multiplexed PCR-Free Detection of MicroRNAs in Single Cancer Cells Using a DNA-Barcoded Microtrough Array Chip
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DOI：
10.3390/mi10040215
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发表时间：
2019-04-01
期刊：
影响因子：
3.4
通讯作者：
Fan, Rong
中科院分区：
文献类型：
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作者：
Wang, Nayi;Lu, Yao;Fan, Rong
MicroRNA是一类小RNA分子，其在广泛的生物过程中调节mRNA的表达，并与人类健康和疾病如癌症有关。如何以高通量测量单细胞中的microRNA谱对于开发用于询问microRNA介导的肿瘤内异质性的基于细胞的测定以及设计用于诊断和监测癌症的新实验室测试至关重要。在这里，我们报告了在亚纳升微槽阵列芯片中实现的原位杂交条形码工作流程，用于在单细胞水平上进行高通量和多重microRNA检测。微槽用于包封固定、透化并与微RNA检测探针预孵育的单细胞，每个微RNA检测探针由与特定微RNA互补的捕获链和独特的报告链组成，该报告链可以在微槽中被光切割，随后通过微槽底部图案化的DNA条形码阵列检测。以这种方式，从单细胞释放的报告链的测量是用于检测单细胞microRNA谱的替代。这种方法允许直接测量microRNA，而无需PCR扩增，因为体积小（
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 (