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一种重要的工具，不同于正常与疾病状态下CNS的全局和区域分析，因为每个神经细胞都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。 所提出的RANP利用数字电极来控制离散的液滴，使得能够在单个芯片上进行RNA分离、cDNA扩增、生物素标记和靶纯化。 结果，可以从来自单个细胞的“亚皮克”mRNA产生“微克”纯化的cDNA。 RANP系统能够在非常小的体积上进行多步（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。在第一阶段的项目中，设计，制造的RANP芯片，并测试cDNA合成，SPIA cDNA扩增，cDNA片段化，和生物素标记。从几皮克的总RNA中获得约500，000倍的扩增。纯化的cDNA在200-4000个核苷酸的范围内，具有约500个碱基的峰，并且没有可检测的残留引物。这产生了248 ng纯化的扩增cDNA，其在我们的I期靶标内。该技术提供了对单细胞转录谱的稳健鉴定的潜力，这是用当前技术无法实现的。 第二阶段的工作将集中在原型RANP芯片的设计和开发，nanodrop芯片的优化，RANP系统的全自动化集成，基于RANP的生物测定的优化，并建立RANP芯片和生物测定规范。

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.