Automated Tag Library Generation for Sequencing-Based Gene Expression Profiling

用于基于测序的基因表达谱的自动标签库生成

• 批准号: 7643949
• 负责人: Gao Chen
• 金额: $ 19.93万
• 依托单位: MAXWELL SENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-25 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7643949
• 关键词: Amplifiers; Automation; Base Sequence; Biochemical Process; Biochemical Reaction; Biological; Biological Assay; California; Cells; Chemistry; Collaborations; Collection; Complementary DNA; Complex; Data; Development; Diagnosis; Digestion; Dimensions; Disease; Electrodes; Environment; Evaluation; Feasibility Studies; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Generations; Genes; Genome; Goals; Heating; Housekeeping Gene; Human; Human Genetics; Incubated; Individual; Kidney; Knowledge; Lead; Libraries; Ligation; Liquid substance; Magnetism; Manuals; Measures; Messenger RNA; Methods; MicroRNAs; Microarray Analysis; Microfluidics; Nucleic Acid Probes; Nucleotides; Operative Surgical Procedures; Pathway interactions; Performance; Persons; Phase; Preparation; Procedures; Process; Protocols documentation; RNA; RNA Processing; RNA Splicing; Reaction; Reading; Reagent; Reproducibility; Research Personnel; Residual state; Reverse Transcription; Risk; Robotics; Running; Sampling; Sequence Analysis; Series; Side; Site; Solutions; Source; Specificity; Structure; Surface; System; Technology; Testing; Time; Tissue Extracts; Tissue-Specific Gene Expression; Transcript; Tube; Universities; Viscosity; Workload; base; cost; design; digital; disease classification; electric field; improved; interfacial; kidney cell; magnetic beads; micro-total analysis system; nanoDroplet; nanolitre; nanosystems; parallel processing; prototype; public health relevance; research facility; research study; sensor; stoichiometry; voltage

DESCRIPTION (provided by applicant): Gene expression profiling, the use of technology to detect cellular transcripts in a high-throughput and massively parallel fashion, has started to move from the solely hybridization-based methods used in microarray technology to sequencing-based technologies for their increased sensitivities (10-100 times) and their ability to quantify virtually all messenger RNAs (mRNAs), including unknown transcripts, alternatively spliced, as well as microRNAs. While some systems exist to handle sample preparation for microarray applications of gene expression, the precursory target preparation steps for sequencing-based gene expression profiling are performed manually. The very tedious procedure requires one skilled full-time person 3 days to perform more than 50 steps, including mRNA isolation, cDNA synthesis and purification, cDNA cleavage and adapter ligation, as well as PCR amplification and target purification. As the procedure involves numerous pipetting steps and reagent transfers, it presents an increased risk of contamination and a high likelihood of manual error accumulation, which in turn creates lower confidence data. Currently there is no system available to automate this tedious workload. Therefore, Maxwell Sensors Inc. (MSI) proposes to develop an Automated Integrated RNA Amplifier (AIRA) chip and system for generating digital cDNA tag libraries from biological samples for sequencing-based gene expression analysis. The proposed AIRA platform combines electrowetting-based nanodroplet manipulation and mRNA amplification chemistry using an integrated labon- a-chip system. The proposed AIRA harnesses the power of electrowetting-based digital fluidics that not only can automate all the sample preparation steps by dispensing, transporting, splitting, merging, and mixing of droplets, but also enables reactions to be performed in discrete nanoliter quantities on a single chip. Tremendous labor and time will be saved. The AIRA offers the potential for robust generation of millions of transcript fragments that can be identified by massively parallel sequencing at a fraction of the current cost. PUBLIC HEALTH RELEVANCE: Automated Integrated RNA Amplifier (AIRA) chip and system will be developed for processing RNA from biological samples in an automated, reliable, and cost-effective manner. The processed RNA can be directly applied to existing cutting-edge sequencing technologies to create a global RNA profile. The system will benefit researchers who are seeking better diagnosis and cures for diverse complex diseases.

描述（由申请人提供）：基因表达谱，即以高通量和大规模并行方式检测细胞转录物的技术，已经开始从微阵列技术中使用的仅基于杂交的方法转向基于测序的技术它们提高了灵敏度（10-100 倍），并且能够量化几乎所有信使 RNA (mRNA)，包括未知转录本、选择性剪接以及 microRNA。虽然存在一些系统来处理基因表达微阵列应用的样品制备，但基于测序的基因表达谱的前期靶标制备步骤是手动执行的。这一繁琐的过程需要一名熟练的全职人员3天时间完成50多个步骤，包括mRNA分离、cDNA合成和纯化、cDNA切割和接头连接、以及PCR扩增和靶标纯化。由于该过程涉及大量移液步骤和试剂转移，因此污染风险增加，并且手动错误累积的可能性很高，进而产生置信度较低的数据。目前还没有系统可以自动完成这项繁琐的工作。因此，Maxwell Sensors Inc. (MSI) 提议开发一种自动集成 RNA 放大器 (AIRA) 芯片和系统，用于从生物样本中生成数字 cDNA 标签库，用于基于测序的基因表达分析。所提出的 AIRA 平台使用集成的 labon-a-chip 系统将基于电润湿的纳米液滴操作和 mRNA 扩增化学相结合。所提出的 AIRA 利用基于电润湿的数字流体技术，不仅可以通过液滴的分配、运输、分裂、合并和混合来自动化所有样品制备步骤，而且还可以在单​​个液滴上以离散纳升量进行反应。芯片。将节省大量的劳动力和时间。 AIRA 提供了稳健生成数百万转录本片段的潜力，这些转录本片段可以通过大规模并行测序来识别，而成本仅为当前成本的一小部分。 公共健康相关性：将开发自动集成 RNA 放大器 (AIRA) 芯片和系统，用于以自动化、可靠且经济高效的方式处理生物样本中的 RNA。处理后的RNA可以直接应用于现有的尖端测序技术，以创建全球RNA谱。该系统将使那些正在寻求更好地诊断和治疗各种复杂疾病的研究人员受益。