High-throughput, Multiplexed Detection of miRNA Biomarkers in Single Cancer Cells

描述(申请人提供)：microRNAs(MiRNAs)是最近发现的一类作为负基因调节因子的非编码小RNA。一大批miRNAs位于染色体的脆弱区域，这是基因组中与人类癌症关系更密切的区域，表明miRNAs在肿瘤发展中可能发挥作用。已有研究表明，miRNA表达谱与多种癌症相关，miRNA特征信号可能是准确诊断和分类人类癌症的唯一癌症生物标志物。然而，人类癌症具有高度的异质性，部分原因是肿瘤内单细胞水平的高度异质性，需要对大量单个肿瘤细胞进行评估，每个细胞的miRNA表达谱都可以测量。在这里，我们建议将单细胞条形码芯片平台与基于连接的新型miRNA检测方案相结合，开发一种创新的技术，首次提供高通量和多路复用，以实现单细胞中不需要扩增和低成本的快速miRNA检测。更具体地说，我们将：(1)设计并验证DNA条码微阵列，用于在不进行扩增的情况下对miRNA生物标志物进行多路检测；(2)将该条码阵列与纳升微流控芯片集成，对大量单个癌细胞中的12个miRNA生物标志物进行多路检测。我们的技术是一个通用的平台，可以检测与各种人类癌症有关的miRNA生物标记物。它可以被进一步修改，以检测单细胞中的其他非编码RNA和信使RNA生物标记物。此外，所提出的微芯片价格低廉、可扩展且易于制造。它可能会立即在基础研究和人类癌症的临床分层中得到应用。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are a class of small non-coding RNAs recently discovered as negative gene regulators. A large faction of miRNAs are located within the fragile regions of chromosomes, which are areas of the genome more tightly associated with human cancers, indicating the possible role of miRNAs in tumor development. It has been shown that miRNA expression profiles correlate with various cancers and the miRNA signatures are potentially unique cancer biomarkers to accurately diagnose and classify human cancers. However, human cancers are highly heterogeneous due in part to the high degree of intratumoral heterogeneity at the single-cell level, requiring the evaluation of a large number of single tumor cells, each of which the miRNA expression profile can be measured. Here we propose to combine a single-cell barcode chip platform and a novel ligation-based miRNA detection scheme to develop an innovative technology that for the first time offers both high-throughput and multiplexing for rapid miRNA detection in single cells without amplification and at low cost. More specifically, we will: (1) Design and validate a DNA barcode microarray for multiplexed detection of miRNA biomarkers without amplification, and (2) Integrate this barcode array with a nanoliter microfluidics chip to perform multiplexed detection of 12 miRNA biomarkers from a large number of single cancer cells. Our technology is a versatile platform to detect miRNA biomarkers implicated in a variety of human cancers. It can be further modified to detect other non-encoding RNAs and messenger RNA biomarkers from single cells. Moreover, the proposed microchip is inexpensive, scalable and easy to manufacture. It may find immediate applications in basic research and clinical stratification of human cancers.