PIXUL-FFPE: High throughput platform for chromatin and DNA sample preparation from formalin fixed paraffin embedded (FFPE) tissue samples.

PIXUL-FFPE：用于从福尔马林固定石蜡包埋 (FFPE) 组织样本中制备染色质和 DNA 样本的高通量平台。

• 批准号: 10021842
• 负责人: Karol Bomsztyk
• 金额: $ 99.52万
• 依托单位: MATCHSTICK TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-18 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021842
• 关键词: Address; Archives; Benchmarking; Biological Assay; Biological Markers; Biological Specimen Banks; Buffers; Chromatin; Chromatin Remodeling Factor; Clinical; Consumption; Custom; DNA; DNA Modification Process; Data; Detection; Devices; Disease; Disease Pathway; Drug Targeting; Engineering; Enzymes; Epigenetic Process; Feedback; Formalin; Freezing; Genes; Genome; Glioblastoma; Goals; Heating; Histologic; Histones; Human; Laboratories; Letters; Market Research; Matched-Pair Analysis; Measures; Medicine; Methods; Molecular; Paraffin Embedding; Pathway interactions; Phase; Polypropylenes; Polystyrenes; Post-Translational Protein Processing; Preparation; Procedures; Process; Proteins; Protocols documentation; RNA; RNA Polymerase II; Recovery; Reproducibility; Research; Research Personnel; Resistance; Resources; Retrieval; Sampling; Series; Signal Transduction; Small Business Technology Transfer Research; Solvents; Specimen; Technology; Temperature; Testing; Time; Tissue Embedding; Tissue Sample; Tube; Ultrasonic Transducer; Ultrasonography; base; biomarker discovery; cancer biomarkers; chromatin immunoprecipitation; comparative; design; epigenetic marker; epigenetic therapy; human tissue; improved; instrument; interest; melting; precision medicine; prototype; repository; tool; tumor; user-friendly

Formalin fixed paraffin-embedded (FFPE) tissue archives represent the largest clinically annotated human specimen repository. The estimated millions of available FFPE tissue samples provide vast resource for the discovery of disease epigenetic pathways, biomarkers and drug targets, And yet, these samples are highly underutilized because current methods for extracting molecular information from FFPE samples are slow, labor intensive, insufficiently sensitive to detect gene-bound enzymes (drug targets) and have low throughput to fully exploit their enormous research and clinical potential. There is an unmet need for better tools to take advantage of these archived clinical samples to retrieve chromatin and DNA for the discovery of epigenetic biomarkers for personal medicine. The chromatin immunoprecipitation (ChIP) assay, a widely used approach for identifying genome- associated proteins or DNA modifications, is one of the most powerful tools used in epigenetic studies. To facilitate chromatin analysis, we have developed a high-throughput microplate-based Matrix ChIP-qPCR platform and engineered a device for fast chromatin shearing which uses an array of 96 miniature ultrasound transducers PIXUL (PIXelated ULtrasound). Extraction of chromatin from FFPE samples is a multistep process which includes chromatin shearing. Ultrasound facilitates chromatin extraction from FFPE samples. The goal of this Matchstick Fast Track STTR project (working with UW partner laboratories) is to develop and validate a user-friendly and efficient high throughout PIXUL-based technology, PIXUL-FFPE, to extract and shear chromatin and DNA from small FFPE samples for epigenetic analysis for use in the research market paving the way for use in clinical settings. The following aims are proposed. In Aim#1(Phase 1) We will design and validate custom PIXUL 96-well microplate for more efficient extraction of chromatin and DNA from FFPE tissue samples. In Aim#2 (Phase 2) we will optimize PIXUL-FFPE protocol for fast and high throughput chromatin extraction from small FFPE samples while maximizing sample integrity. In Aim#3 (Phase 2) we will demonstrate the translational utility of PIXUL-FFPE by comparative ChIP analysis of matched pairs of fresh-frozen and FFPE glioblastoma (GBM) tumor sections. In Aim#4 (Phase 2) we will modify PIXUL instrument for extraction of chromatin from FFPE samples. Search for epigenetic therapies has slowed down by paucity of available data. Thus, integrating the custom 96-well plates and improved protocol with the modified PIXUL instrument will greatly increase utilization of FFPE specimens in the research market place attracting greater numbers of investigators to explore the vast repositories of archived human tissues and as such advancing precision medicine.

福尔马林固定石蜡包埋 (FFPE) 组织档案代表了最大的临床注释人类 标本库。估计有数百万可用的 FFPE 组织样本为研究提供了巨大的资源。 疾病表观遗传途径、生物标志物和药物靶标的发现，然而，这些样本高度 由于当前从 FFPE 样品中提取分子信息的方法速度慢、费力，因此未得到充分利用 强度大，检测基因结合酶（药物靶标）不够灵敏，并且通量低，无法充分检测 充分利用其巨大的研究和临床潜力。对更好的工具的需求尚未得到满足 对这些存档的临床样本进行检索，以检索染色质和 DNA，以发现表观遗传生物标志物 个人用药。 染色质免疫沉淀 (ChIP) 测定是一种广泛使用的基因组识别方法 相关蛋白质或 DNA 修饰是表观遗传学研究中最强大的工具之一。到 为了促进染色质分析，我们开发了基于微孔板的高通量 Matrix ChIP-qPCR 平台并设计了一种用于快速染色质剪切的装置，该装置使用 96 个微型超声波阵列 传感器 PIXUL（PIXelated ULtrasound）。从 FFPE 样品中提取染色质是一个多步骤过程 其中包括染色质剪切。超声波有助于从 FFPE 样品中提取染色质。目标是 这个火柴棒快速通道 STTR 项目（与华盛顿大学合作伙伴实验室合作）旨在开发和 验证用户友好且高效的基于 PIXUL 的高通量技术 PIXUL-FFPE，以提取 从小 FFPE 样品中剪切染色质和 DNA，进行表观遗传分析以供研究使用 市场为临床应用铺平道路。提出以下目标。在目标#1（第一阶段）中，我们 将设计和验证定制 PIXUL 96 孔微孔板，以更有效地提取染色质和 DNA 来自 FFPE 组织样本。在 Aim#2（第 2 阶段）中，我们将优化 PIXUL-FFPE 协议，以实现快速、高效 从小型 FFPE 样品中进行高通量染色质提取，同时最大限度地提高样品完整性。目标#3（阶段 2) 我们将通过配对的 ChIP 比较分析来展示 PIXUL-FFPE 的转化效用 新鲜冷冻和 FFPE 胶质母细胞瘤 (GBM) 肿瘤切片。在 Aim#4（第 2 阶段）中，我们将修改 PIXUL 仪器 用于从 FFPE 样品中提取染色质。 由于缺乏可用数据，对表观遗传疗法的搜索已经放缓。因此，整合定制 96 孔板和改进的 PIXUL 仪器改进方案将大大提高 FFPE 的利用率 研究市场上的标本吸引了更多的研究人员探索广阔的领域 存档人体组织的存储库，从而推进精准医学。