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（由申请人提供）：中风是美国第三大杀手，也是每年超过 795,000 例成人残疾的主要原因。中风的两种主要类型：缺血性和出血性，在临床上无法区分。 30% 出现类似中风症状的患者根本没有中风。快速准确地做出中风诊断势在必行，因为缺血性和出血性中风需要不同的治疗方法，而这些治疗方法的有效时间窗口很短（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，这会延迟治疗干预。不幸的是，目前尚不存在针对中风的分子诊断测试，主要是因为潜在的分子标记物组合正在等待临床验证。该验证过程在一定程度上受到分析所用工具的阻碍，例如用于核酸标记物的逆转录 PCR (RT-PCR) 或用于血清蛋白标记物的免疫测定，因为缺乏样本处理自动化且使用了冗长的处理步骤。在此应用中，将开发一种高度创新的全自动系统，该系统具有精湛的分析灵敏度，可以监测不同分子标记表达水平的微小变化，周转时间 (TAT) 小于 15 分钟。该系统将有助于识别和验证新的分子标记，以实现快速、特异和灵敏的中风诊断。分子测定和新型硬件将用于评估信使 RNA 标记物组作为前所未有的基于血液的测试（使用外周血单核细胞，PBMC）用于中风诊断的潜力。两项技术将构成该系统的核心：（1）由聚合物通过复制制成的模块化流体生物处理器，包含所有样品处理步骤； (2) 单对荧光共振能量转移 (spFRET)，可消除多个样品处理步骤。微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所需的一次性应用。该生物处理器将包含一个流体母板，其上互连有特定任务的模块，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)反转录成cDNA； (5) 连接酶检测反应（LDR）以生成具有供体/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）计数将能够消除多个样品预处理步骤，从而缩短测定 TAT，并产生必要的分析灵敏度来检测 mRNA 标记物表达水平的细微变化，这在中风事件后的早期可能至关重要。该系统可用于任何需要 mRNA 表达谱分析的应用，但将在当前项目中用于验证中风诊断的分子标记。