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

描述（由申请人提供）：微小RNA（miRNAs）是最近发现的一类小的非编码RNA，作为负基因调节因子。大部分miRNA位于染色体的脆弱区域内，这些区域是与人类癌症更紧密相关的基因组区域，表明miRNA在肿瘤发展中的可能作用。研究表明，miRNA的表达谱与多种癌症相关，miRNA标志物是一种潜在的独特的癌症生物标志物，可用于人类癌症的准确诊断和分类。然而，人类癌症是高度异质性的，部分原因是在单细胞水平上肿瘤内的高度异质性，需要评估大量的单个肿瘤细胞，每个肿瘤细胞的miRNA表达谱都可以测量。在这里，我们提出了联合收割机结合单细胞条形码芯片平台和一种新的基于连接的miRNA检测方案，以开发一种创新技术，首次提供高通量和多路复用，用于在单细胞中快速检测miRNA，而无需扩增，成本低。具体而言，我们将：（1）设计并验证用于多重检测miRNA生物标志物而不扩增的DNA条形码微阵列，以及（2）将该条形码阵列与纳升微流体芯片整合以执行来自大量单个癌细胞的12种miRNA生物标志物的多重检测。我们的技术是一个多功能的平台，可以检测与各种人类癌症有关的miRNA生物标志物。它可以被进一步修饰以检测来自单细胞的其他非编码RNA和信使RNA生物标志物。此外，所提出的微芯片是廉价的，可扩展的，易于制造。它可能会立即应用于人类癌症的基础研究和临床分层。

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.