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）相关疾病是人类最常见的死亡和降低生活质量的原因之一。在理解和对抗中枢神经系统相关疾病的努力中，分析单个神经细胞的能力是一个重要的工具，不同于对正常和疾病状态下中枢神经系统的整体和区域分析，因为每个神经细胞都有独特的分子特征。然而，由于单细胞中信使RNA （mRNA）含量低，单细胞基因表达谱分析目前受到阻碍。从单个细胞中收获的mRNA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，并且可能在扩增前过程中丢失。此外，由于mRNA模板的稀释和酶效率的降低，它经常导致有偏差的数据，从而影响生物解释。因此，Maxwell Sensors公司建议开发一种RNA扩增纳米处理器（RANP），用于并行处理来自单个哺乳动物细胞的全局mRNA。提出的RANP利用数字电极来控制离散液滴，使RNA分离，cDNA扩增，生物素标记和目标纯化在单个芯片上。结果，纯化cDNA的“微图”可以从单细胞的“亚图”mRNA中产生。RANP系统可以在非常小的体积上自动处理多步骤（20-25步）反应，因此样品加载是唯一的手动步骤。在一期项目中，ranp芯片被设计、制造并测试用于cDNA合成、SPIA cDNA扩增、cDNA片段化和生物素标记。从总RNA的几个图中获得了大约50万倍的扩增。纯化的cDNA在200-4000个核苷酸之间，峰值在500个碱基左右，没有检测到残留的引物。这产生了248ng纯化的扩增cDNA，符合我们第一阶段的目标。这项技术为单细胞转录谱的可靠鉴定提供了潜力，这是目前技术无法实现的。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，以实现全面自动化、基于RANP的生物测定的优化，并建立RANP芯片和生物测定规范。