spFRET for Expression Profiling mRNA: Discovering Markers for Stroke Diagnosis

spFRET 用于 mRNA 表达谱分析：发现中风诊断标记物

• 批准号: 8427393
• 负责人: Alison E Baird
• 金额: $ 62.9万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8427393
• 关键词: Adult; Architecture; Automation; Biochemical Process; Biological Assay; Biological Markers; Blood; Blood Platelets; Brain hemorrhage; Cell Fraction; Cell Separation; Cells; Clinical; Clinical Data; Clinical Research; Clinical Sensitivity; Complementary DNA; Coupled; Cytolysis; Data Collection; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Dimensions; Enzymes; Erythrocytes; Event; Extravasation; Fiber Optics; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Hour; Housekeeping Gene; Image; Immunoassay; In Vitro; Ischemic Stroke; Ligase; Light; Liquid substance; Magnetic Resonance Imaging; Messenger RNA; Microfluidics; Molecular; Molecular Diagnostic Testing; Molecular Profiling; Monitor; Nucleic Acids; Optics; Output; Patients; Performance; Peripheral; Phase; Photons; Pilot Projects; Poly T; Polymers; Process; RNA marker; Reaction; Reaction Time; Reader; Reverse Transcription; Sampling; Secure; Sensitivity and Specificity; Series; Serum Proteins; Signal Transduction; Solid; Stroke; Symptoms; System; Technology; Testing; Therapeutic Intervention; Time; Validation; Whole Blood; X-Ray Computed Tomography; base; cost; design; digital; disability; flexibility; innovation; innovative technologies; kinematics; mRNA Expression; molecular marker; neutrophil; new technology; novel; novel marker; optical fiber; peripheral blood; prevent; prototype; public health relevance; seal; single molecule; submicron; tool

DESCRIPTION (provided by applicant): Stroke is the third leading killer in the US and the main cause for over 795,000 cases of adult disability each year. The two major types of stroke, ischemic and hemorrhagic, cannot be clinically differentiated; 30% of patients presenting stroke-like symptoms do not have a stroke at all. It is imperative that a stroke diagnosis be made quickly and accurately because ischemic and hemorrhagic strokes require different treatments that have only a small window of time to be effective (3-6 hours). Computed tomography (CT) or magnetic resonance imaging (MRI) is commonly used for the diagnosis, which delays therapeutic intervention. Unfortunately, a molecular diagnostic test does not currently exist for stroke due primarily to the fact that potential molecular marker panels are waiting to be clinically validated. This validation process is, in part, hampered by the tools used for their analysis, such as reverse-transcription PCR (RT-PCR) for nucleic acid-based markers or immunoassays for serum protein markers, due to the lack of sample process automation and the lengthy processing steps used. In this application, a highly innovative, fully-automated system will be developed that has exquisite analytical sensitivity to monitor minute changes in the expression levels of different molecular markers with a turn-around-time (TAT) of less than 15 minutes. The system will facilitate the identification and validation of new molecular markers for the rapid, specific, and sensitive diagnosis of stroke. The molecular assay and novel hardware will be used to evaluate the potential of a messenger RNA marker panel as an unprecedented blood-based test (using peripheral blood mononucleated cells, PBMC) for the diagnosis of stroke. Two technologies will form the core of the system: (1) modular fluidic bio-processor made from polymers via replication that contains all of the sample processing steps; and (2) single pair fluorescence resonance energy transfer (spFRET) that eliminates several sample processing steps. Micro-replication will be used for the fabrication of the bio-processor to keep cost low and make this consumable appropriate for single- use applications, as demanded by clinical diagnostics. The bio-processor will contain a fluidic motherboard with task-specific modules interconnected on it to provide design flexibility. The molecular processing steps poised on the processor will be: (1) PBMC isolation; (2) PBMC lysis; (3) mRNA solid-phase purification; (4) reverse transcription to cDNA; (5) ligase detection reactions (LDRs) to generate molecular beacons with donor/acceptor fluorescent pairs and; (6) spFRET digital detection. The molecular (i.e., digital) counting using spFRET will provide the ability to eliminate several sample pre-processing steps shortening the assay TAT and generate the necessary analytical sensitivity to detect subtle changes in expression levels of mRNA markers, which may be critical during early times following a stroke event. The system can potentially be used for any application requiring mRNA expression profiling, but will be used in the current project for validating molecular markers for the diagnosis of stroke.

描述(申请人提供)：中风是美国第三大杀手，每年超过795,000例成人残疾的主要原因。两种主要的中风类型，缺血性和出血性，在临床上无法区分；30%出现中风样症状的患者根本没有中风。必须快速准确地作出中风诊断，因为缺血性中风和出血性中风需要不同的治疗方法，而这些治疗方法只有很小的时间窗才能奏效(3-6小时)。计算机断层扫描(CT)或磁共振成像(MRI)通常用于诊断，这推迟了治疗干预。不幸的是，目前还不存在中风的分子诊断测试，这主要是因为潜在的分子标记板正在等待临床验证。这一验证过程在一定程度上受到用于其分析的工具的阻碍，例如核酸标记物的逆转录聚合酶链式反应(RT-PCR)或血清蛋白质标记物的免疫分析，这是由于缺乏样品处理自动化和使用的漫长的处理步骤。在这项应用中，将开发一种高度创新的全自动化系统，该系统具有精致的分析灵敏度，可以监测不同分子标记表达水平的微小变化，周转时间(TAT)不到15分钟。该系统将有助于识别和验证新的分子标记，以快速、特异和敏感地诊断中风。分子分析和新的硬件将被用于评估信使RNA标记板作为一种史无前例的基于血液的检测(使用外周血单核细胞)诊断中风的潜力。两项技术将构成该系统的核心：(1)通过复制由聚合物制成的模块化射流生物处理器，它包含所有样品处理步骤；以及(2)单对荧光共振能量转移(SpFRET)，它省去了几个样品处理步骤。微型复制将用于生物处理器的制造，以保持低成本，并使该消耗品适合临床诊断要求的一次性应用。生物处理器将包含一个流体主板，主板上互连着特定于任务的模块，以提供设计灵活性。处理器上的分子处理步骤将是：(1)PBMC分离；(2)PBMC裂解；(3)mRNA固相纯化；(4)反转录为cDNA；(5)连接酶检测反应(LDRs)，以产生带有供体/受体荧光对的分子信标；(6)spFRET数字检测。使用spFRET的分子(即数字)计数将提供省去几个样品前处理步骤的能力，从而缩短分析TAT并产生必要的分析灵敏度，以检测在中风事件后的早期时间内可能是关键的mRNA标记物表达水平的细微变化。该系统潜在地可以用于任何需要mRNA表达谱的应用，但将在当前的项目中用于验证中风诊断的分子标记。