Single Cell RNA Amplification Nanodrop Processor

单细胞 RNA 扩增 Nanodrop 处理器

• 批准号: 7388346
• 负责人: Gao Chen
• 金额: $ 15.62万
• 依托单位: MAXWELL SENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-15 至 2009-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7388346
• 关键词: Affect; Automation; Biochemical Reaction; Biological; Biological Assay; Biotin; California; Cells; Cessation of life; Chemistry; Collection; Complementary DNA; Computers; Condition; Cytolysis; DNA amplification; Data; Dimensions; Disease; Electrodes; Environment; Figs - dietary; Formalin; Gene Expression Profiling; Harvest; Heating; Human; Label; Liquid substance; Magnetism; Mammalian Cell; Messenger RNA; Microprocessor; Molecular Profiling; Nerve; Neurons; Nucleic Acid Probes; Paraffin; Procedures; Process; Quality of life; RNA; RNA Processing; RNA amplification; Reaction; Reagent; Sampling; Side; Site; Solutions; Standards of Weights and Measures; Surface; System; Technology; Viscosity; micro-total analysis system; nanoDroplet; nanolitre; parallel processing; sensor; stoichiometry

DESCRIPTION (provided by applicant): Central nerve system (CNS) related diseases are one of the most prevalent causatives of death and reduced life quality among human beings. In an effort to understand and combat CNS-related diseases, the ability to analyze single neural cells is an important distinction from global and regional analyses of the CNS in the state of normal function versus disease, as each neural cell has a unique molecular signature. However, single-cell gene expression profiling is currently hampered due to the low amount of messenger RNA (mRNA) present in a single cell. The quantity of mRNA harvested from a single cell, estimated to be approximately 0.1-0.2 picograms, is below the level of sensitivity for standard RNA extraction procedures and is likely to have losses during the preamplification processes. Furthermore, because of the dilution of the mRNA templates and reduction in enzymatic efficiency, it often results in biased data, which affects biological interpretation. Therefore, Maxwell Sensors Inc. proposes to develop an RNA Amplification Nanodrop Processor (RANP) for parallel processing of global mRNA from single mammalian cells. The proposed RANP platform combines three technologies: nanodroplet manipulation, electro-wetting, and mRNA extraction/amplification chemistry into an integrated lab-on-a-chip system. Nanodrop manipulation enables reactions to be performed in a discrete nanoliter quantity, while electro-wetting offers the direct liquid manipulation, including dispensing, transporting, splitting, merging, and mixing of droplets. In combination of these technologies, the RANP system will automate cell lysis, RNA isolation, DNA amplification, biotin labeling, and target purification from a single cell. The resulting system will minimize loss of mRNA template through automation of all steps of RNA processing, minimize stochastic effects by controlled manipulation of nanoliter volumes, process hundreds of single cells simultaneously on a single chip, and increase the consistency and reliability of downstream microarray assays for single-cell profiling. It offers the potential to robustly identify a single cell's transcriptional profile, which is not able to be achieved with current technologies.

描述（由申请人提供）：中枢神经系统（CNS）相关疾病是人类死亡和生活质量下降的最常见原因之一。为了理解和对抗中枢神经系统相关疾病，分析单个神经细胞的能力是对中枢神经系统在正常功能与疾病状态下的全局和区域分析的一个重要区别，因为每个神经细胞都有独特的分子特征。然而，由于单细胞中存在的信使 RNA (mRNA) 含量较低，单细胞基因表达谱目前受到阻碍。从单个细胞中收获的 mRNA 数量估计约为 0.1-0.2 皮克，低于标准 RNA 提取程序的灵敏度水平，并且可能在预扩增过程中发生损失。此外，由于 mRNA 模板的稀释和酶效率的降低，通常会导致数据出现偏差，从而影响生物学解释。 因此，Maxwell Sensors Inc. 提议开发一种 RNA 扩增 Nanodrop 处理器 (RANP)，用于并行处理来自单个哺乳动物细胞的全局 mRNA。拟议的 RANP 平台将三种技术结合在一起：纳米液滴操作、电润湿和 mRNA 提取/扩增化学，形成集成的芯片实验室系统。纳米液滴操作使反应能够以离散的纳升量进行，而电润湿提供直接的液体操作，包括液滴的分配、运输、分裂、合并和混合。结合这些技术，RANP 系统将自动进行细胞裂解、RNA 分离、DNA 扩增、生物素标记和单细胞靶标纯化。由此产生的系统将通过RNA处理所有步骤的自动化最大限度地减少mRNA模板的损失，通过纳升体积的控制操作最大限度地减少随机效应，在单个芯片上同时处理数百个单细胞，并提高下游单细胞分析微阵列分析的一致性和可靠性。它提供了可靠地识别单个细胞的转录谱的潜力，这是当前技术无法实现的。