Single Cell RNA Amplification Nanodrop Processor

描述(申请人提供)：中枢神经系统(CNS)相关疾病是导致人类死亡和生活质量下降的最常见原因之一。在努力了解和抗击中枢神经系统相关疾病的过程中，分析单个神经细胞的能力是一种重要的工具，不同于对正常和疾病状态下的中枢神经系统的全局和区域性分析，因为每个神经细胞都有一个独特的分子特征。然而，单细胞基因表达谱目前受到阻碍，因为在单个细胞中存在低数量的信使RNA(MRNA)。从单个细胞中获取的信使核糖核酸的数量估计约为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，往往会导致数据的偏差，从而影响生物学解释。因此，Maxwell Sensors Inc.建议开发一种RNA扩增纳米滴处理器(RANP)，用于并行处理单个哺乳动物细胞的全局mRNA。所提出的RANP利用数字电极来控制离散的液滴，从而使RNA分离、cDNA扩增、生物素标记和目标纯化能够在单个芯片上进行。其结果是，从单个细胞中提取的信使核糖核酸“亚皮克”即可产生“微克”的纯化基因。RANP系统能够在很小的体积上实现多步骤(20-25步)反应的自动化处理，因此样品加载是唯一的手动步骤。在第一阶段项目中，设计、制造了RANP-芯片，并对其进行了cDNA合成、SPIA cDNA扩增、cDNA片段和生物素标记的测试。从几个皮克的总RNA中可以获得大约50万倍的扩增。纯化后的cDNA量在200-4000个核苷酸之间，峰在500个碱基左右，没有检测到残留的引物。这产生了248 ng的纯化扩增的cdna，这在我们的第一阶段目标内。这项技术提供了强大的识别单个细胞的转录图谱的潜力，这是目前的技术无法实现的。第二阶段的工作将集中于RANP芯片原型的设计和开发、纳米滴芯片的优化、RANP系统的集成以实现全自动化、基于RANP的生物检测的优化，以及建立RANP芯片和生物检测规范。

DESCRIPTION (provided by applicant): Central nerve system (CNS) related diseases are one of the most prevalent causes of death and reduced life quality among human beings. In the effort to understand and combat CNS-related diseases, the ability to analyze single neural cells is an important tool distinct from global and regional analyses of the CNS in normal versus disease states, 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 utilizes digital electrodes to control discrete droplets that enable RNA isolation, cDNA amplification, biotin labeling, and target purification on a single chip. As the result, "microgram" of purified cDNA can be generated from "sub- picogram" of mRNA from a single cell. The RANP system enables automated processing on a very small volume for multi-step (20-25 steps) reactions, so that sample loading is the only manual step. During the Phase I project, the RANP-chip was designed, fabricated, and tested for cDNA synthesis, SPIA cDNA amplification, cDNA fragmentation, and biotin labeling. Approximately ~500,000-fold amplification was obtained from a few picogram of total RNA. The purified cDNA was in the range of 200-4000 nucleotides, with a peak around 500 bases and no detectable residual primers. This generated 248ng of purified amplified cDNA which is within our Phase I target. This technology offers the potential for robust identification of a single cell's transcriptional profile, which cannot be achieved with current technologies. Phase II work will focus on design and development of a prototype RANP chip, optimization of the nanodrop chip, integration of the RANP system for total automation, optimization of the RANP-based bioassay, and establish the RANP-chip and bioassay specifications.