Nanobind FFPE DNA/RNA Extraction

Nanobind FFPE DNA/RNA 提取

• 批准号: 8755436
• 负责人: Kelvin Liu
• 金额: $ 22.43万
• 依托单位: CIRCULOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8755436
• 关键词: Archives; Binding; Biological Assay; Biological Markers; Biological Preservation; Buffers; Capillary Electrophoresis; Cells; Centrifugation; Chloroform; Clinical; Cytolysis; DNA; DNA Binding; Deposition; Diagnosis; Digestion; Electrophoresis; Epidemiologic Studies; Esophageal; Evaluation; Failure; Film; Formaldehyde; Formalin; Freezing; Gold; Heating; Hospitals; Malignant Neoplasms; Medical; Methods; Methylation; MicroRNAs; Molecular; Molecular Analysis; Molecular Profiling; Mutation; Outcome; Paraffin; Paraffin Embedding; Pathologic; Pathology; Patients; Performance; Phase; Phenols; PicoGreen; Process; Protocols documentation; RNA; RNA Degradation; Reproducibility; Research Personnel; Retrospective Studies; Sampling; Scientist; Silicon Dioxide; Small RNA; Solid; Speed; Staining method; Stains; Techniques; Temperature; Tissue Sample; Tissues; Tube; Waxes; Work; base; clinical application; cohort; crosslink; design; empowered; experience; nanomaterials; nanoscale; new technology; novel; polyolefin; public health relevance; sample fixation; tissue processing

DESCRIPTION (provided by applicant): Nanobind FFPE DNA/RNA Extraction Despite the growing need for and the demonstrated potential advantages of molecular biomarkers, it has proven difficult to routinely employ them in the diagnosis and management of patients. One reason for this failure has been the logistical challenges of obtaining, rapidly processing, storin, and transporting quick-frozen tissue samples in clinical settings. Standard hospital tissue processing involves fixation in formaldehyde, followed by embedding in paraffin blocks to generate Formalin Fixed Paraffin Embedded (FFPE) tissue samples. If these FFPE samples can be harnessed for routine molecular analysis, the potential for revolutionizing current medical practice exists. FFPE blocks obtained in hospital pathology departments could then be routinely assayed using the newer molecular methods. Moreover, since FFPE samples are usually stored for many years by hospital pathology departments, retrospective molecular evaluations could also be performed, empowering researchers to conduct molecular epidemiologic studies on large cohorts with known clinical outcomes. We aim to develop a new DNA/RNA FFPE extraction method based on a novel and inexpensively fabricated nanomaterial called Nanobind. Nanobind is a thermoplastic substrate containing a hierarchical topography of microscale folds and nanoscale silica flakes. Unlike beads and columns which impart DNA/RNA fragmenting shear forces, the non-porous Nanobind substrate can bind and release DNA/RNA without fragmenting it, achieving DNA/RNA integrity (>48 kbp) that matches gold standard phenol- chloroform extractions with a process that is simpler than beads and columns (e.g. no magnets, high speed centrifugation, or tube transfers). Furthermore, Nanobind has a binding capacity that is 5 - 30 folds greater than beads and columns. Thus, the ability of Nanobind to achieve high DNA integrity combined with its higher extraction efficiency and its ability to load significantly more tissue into a single extraction, could serve to greatly increase molecular assay sensitivity and reproducibility (i.e. more DNA of higher quality). In Aim 1, we will develop a method to extract high integrity, high yield, and RNA-free DNA from FFPE samples using the Nanobind substrate. In Aim 2, we will develop a modified extraction protocol to extract DNA-free, total RNA from FFPE samples. In Aim 3, we will validate the suitability of the Nanobind extracted DNA/RNA for molecular profiling by performing methylation, mutation, and microRNA analysis using qMSP, qPCR, and RT-qPCR and comparing to commercial column extracted DNA/RNA.

描述（由申请人提供）：尽管需求越来越大，并且表现出了分子生物标志物的潜在优势，但纳米宾FFPE DNA/RNA提取，但事实证明，很难将它们定期利用它们来诊断和管理。发生这种失败的原因之一是在临床环境中获得，快速加工，storin和运输快速冻结的组织样品的后勤挑战。标准的医院组织加工涉及甲醛固定，然后嵌入石蜡块中以产生福尔马林固定的石蜡嵌入（FFPE）组织样品。如果可以利用这些FFPE样品进行常规的分子分析，则存在革新当前医学实践的潜力。然后，可以使用较新的分子方法常规测定在医院病理部门获得的FFPE块。此外，由于医院病理学部门通常将FFPE样品存储多年，因此还可以进行回顾性分子评估，从而使研究人员对具有已知临床结果的大型同类群进行分子流行病学研究。我们旨在开发一种新的DNA/RNA FFPE提取方法，基于一种新型且廉价地制造的纳米材料。 Nanobind是一种热塑性底物，其中包含微观褶皱和纳米级硅片的分层地形。与散发DNA/RNA碎片剪切力的珠子和色谱柱不同，非孔纳米植物底物可以粘合并释放DNA/RNA而不碎片，从而实现DNA/RNA完整性（> 48 kbp）（> 48 kbp），与金色标准的酚类提取物相匹配，而不是比比伯爵夫人（E.G. e.G. geb。传输）。此外，纳米宾的结合能力比珠子和柱高5-30倍。因此，纳米素达到高DNA完整性的能力以及其较高的提取效率以及其将组织显着加载到单个提取中的能力，可以极大地提高分子测定敏感性和可重复性（即更高质量的DNA）。在AIM 1中，我们将开发一种使用Nanobind底物从FFPE样品中提取高完整性，高产量和无RNA DNA的方法。在AIM 2中，我们将开发一种修改的提取方案，以从FFPE样品中提取无DNA的总RNA。在AIM 3中，我们将通过使用QMSP，QPCR和RT-QPCR进行甲基化，突变和MicroRNA分析来验证纳米素提取的DNA/RNA对分子分析的适用性，并与商业柱提取的DNA/RNA进行比较。