Single Cell RNA Amplification Nanodrop Processor

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。在理解和对抗CNS相关疾病的努力中，分析单个神经细胞的能力是与正常功能与疾病状态下CNS的全局和区域分析的重要区别，因为每个神经细胞具有独特的分子特征。然而，单细胞基因表达谱目前由于单个细胞中存在的信使RNA（mRNA）的量低而受到阻碍。从单个细胞中收获的mRNA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，并且在预扩增过程中可能有损失。此外，由于mRNA模板的稀释和酶促效率的降低，它经常导致有偏差的数据，这影响了生物学解释。 因此，麦克斯韦传感器公司提出开发RNA扩增纳米滴处理器（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平台结合了三种技术：纳米液滴操作，电润湿和mRNA提取/扩增化学到一个集成的芯片实验室系统。Nanodrop操作使得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系统将自动化细胞裂解，RNA分离，DNA扩增，生物素标记和单细胞靶向纯化。由此产生的系统将通过RNA处理的所有步骤的自动化来最小化mRNA模板的损失，通过纳升体积的受控操作来最小化随机效应，在单个芯片上同时处理数百个单细胞，并增加下游微阵列测定的一致性和可靠性用于单细胞分析。它提供了强大的识别单细胞转录谱的潜力，这是目前技术无法实现的。

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.