Single Cell RNA Amplification Nanodrop Processor

单细胞 RNA 扩增 Nanodrop 处理器

• 批准号: 7928754
• 负责人: Gao Chen
• 金额: $ 38.75万
• 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 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7928754
• 关键词: Address; Affect; Alzheimer' s Disease; Automation; Automobile Driving; Biological; Biological Assay; Biotin; Blood Vessels; Brain; Buffers; Cause of Death; Cell Count; Cell Extracts; Cell physiology; Cells; Chemistry; Complementary DNA; Complex; Computer software; Cytolysis; DNA Microarray Chip; DNA Primers; Data; Deoxyribonucleases; Deposition; Development; Dimensions; Disease; Dopamine; Electrodes; Electronics; Environment; Enzymes; Epithelial; Ethanol; Eukaryotic Cell; Evaluation; Feasibility Studies; Figs - dietary; Formalin; Foundations; Freezing; Frequencies; Gene Chips; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Generations; Genes; Genetic; Genetic Transcription; Genome; Goals; Harvest; Healthcare Systems; Heating; Human; Human Genome; Hydrogen Peroxide; In Vitro; Incidence; Incubated; Individual; Knowledge; Label; Lead; Liquid substance; Magnetism; Mammalian Cell; Manuals; Masks; Measurement; Measures; Messenger RNA; Methods; Microarray Analysis; Microfluidics; Microprocessor; Microtubule-Associated Protein 2; Molecular Profiling; Monitor; Movement; Nerve; Nervous system structure; Neurodegenerative Disorders; Neuroglia; Neurons; Normal tissue morphology; Nucleic Acid Probes; Nucleic Acids; Nucleotides; Oxidative Stress; Paraffin Embedding; Parkinson Disease; Pattern; Performance; Phase; Phase II Clinical Trials; Phosphate Buffer; Physiologic pulse; Physiological; Population; Printing; Procedures; Process; Production; Protocols documentation; Pump; Quality of life; RNA; RNA amplification; Reaction; Reagent; Reporting; Reproducibility; Research Personnel; Residual state; Resolution; Reverse Transcription; Saline; Sampling; Sensitivity and Specificity; Site; Slide; Small Business Innovation Research Grant; Solid; Solutions; Spinal cord injury; Spottings; Staging; Stroke; Structure; Surface; System; Technology; Temperature; Testing; Tissues; Trauma; Undifferentiated; Work; base; biobank; brain tissue; cell type; combat; computer monitor; design; digital; electric field; innovation; interfacial; magnetic beads; micro-total analysis system; nanoDroplet; nanolitre; neuropsychiatry; operation; parallel processing; phase 1 study; protocol development; prototype; public health relevance; relating to nervous system; research and development; research study; sensor; single cell analysis; tool; voltage; wasting

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.

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工具，有别于正常状态与疾病状态下中枢神经系统的全局和区域分析，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 利用数字电极来控制离散液滴，从而在单个芯片上实现 RNA 分离、cDNA 扩增、生物素标记和目标纯化。 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自动处理多步骤（20-25 步骤）反应，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，并测试了 cDNA 合成、SPIA cDNA 扩增、cDNA 片段化和生物素标记。从几皮克的总 RNA 中获得了约 500,000 倍的扩增。纯化的cDNA在200-4000个核苷酸范围内，峰值在500个碱基左右，并且没有可检测到的残留引物。这产生了 248ng 纯化的扩增 cDNA，其在我们的 I 期目标范围内。该技术提供了对单细胞转录谱进行可靠识别的潜力，这是当前技术无法实现的。 第二阶段工作将重点是设计和开发原型 RANP 芯片、优化 nanodrop 芯片、集成 RANP 系统以实现完全自动化、优化基于 RANP 的生物测定，并建立 RANP 芯片和生物测定规范。