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

单癌细胞中 miRNA 生物标志物的高通量多重检测

• 批准号: 8726351
• 负责人: Patrick S Doyle
• 金额: $ 22.11万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8726351
• 关键词: Address; Advanced Development; Area; Basic Science; Biological Assay; Biological Markers; Biopsy; Buffers; Cancer Biology; Cell Line; Cells; Cellular biology; Chromosomes; Clinical; Collaborations; Complex; Cytolysis; DNA; DNA Sequence; Data Set; Detection; Development; Diagnosis; Differential Diagnosis; Evaluation; Functional RNA; Gene Expression; Gene Expression Regulation; Genome; Goals; Heterogeneity; Human; Immune; Ligation; Malignant Neoplasms; Measures; Messenger RNA; Metric; MicroRNAs; Microfluidics; Modeling; Molecular Profiling; Motivation; Nature; Neurosurgeon; Patients; Performance; Phase; Play; Primary Neoplasm; Process; Protein Analysis; Public Health; Regulator Genes; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Scheme; Specimen; Stratification; System; Technology; Time; Validation; Variant; anticancer research; base; cancer cell; cancer diagnosis; cancer genetics; clinical Diagnosis; commercialization; cost; density; design; innovative technologies; microchip; nanolitre; neoplastic cell; novel; public health relevance; research study; single cell proteins; stem cell differentiation; success; technology development; tool; tumor

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.

描述（申请人提供）：MicroRNA（miRNA）是最近发现的一类小非编码RNA，可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关系更紧密的基因组区域，表明 miRNA 在肿瘤发展中可能发挥作用。研究表明，miRNA 表达谱与各种癌症相关，并且 miRNA 特征是潜在的独特癌症生物标志物，可准确诊断和分类人类癌症。然而，人类癌症具有高度异质性，部分原因是单细胞水平上瘤内异质性很高，需要评估大量单个肿瘤细胞，并且可以测量每个肿瘤细胞的 miRNA 表达谱。在这里，我们建议将单细胞条形码芯片平台和新型基于连接的miRNA检测方案相结合，开发一种创新技术，该技术首次提供高通量和多重技术，用于单细胞中的快速miRNA检测，无需扩增且成本低廉。更具体地说，我们将：(1) 设计和验证 DNA 条形码微阵列，用于多重检测 miRNA 生物标志物而无需扩增，(2) 将此条形码阵列与纳升微流控芯片集成，以对来自大量单个癌细胞的 12 种 miRNA 生物标志物进行多重检测。我们的技术是一个多功能平台，用于检测与多种人类癌症有关的 miRNA 生物标志物。它可以进一步修改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，所提出的微芯片价格低廉、可扩展且易于制造。它可能会立即应用于人类癌症的基础研究和临床分层。