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

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 标本储存库。据估计，数以百万计的FFPE组织样本为 疾病表观遗传途径、生物标记物和药物靶点的发现，然而，这些样本高度 未得到充分利用，因为目前从FFPE样本中提取分子信息的方法速度慢，劳动力 密集，对检测基因结合酶(药物靶标)不够敏感，并且吞吐量较低，无法完全 开发他们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，以利用 从这些存档的临床样本中提取染色质和DNA，以发现表观遗传生物标记物 个人用药。 染色质免疫沉淀(ChIP)分析是一种广泛使用的鉴定基因组的方法- 相关蛋白质或DNA修饰是表观遗传学研究中使用的最强大的工具之一。至 为了便于染色质分析，我们开发了一种高通量的基于微孔板的矩阵芯片-qPCR 平台，并设计了一种使用96个微型超声波阵列的快速染色质剪切设备 换能器PIXUL(像素化超声)。从FFPE样品中提取染色质是一个多步骤的过程 包括染色质剪切。超声波有助于从FFPE样品中提取染色质。的目标是 该Matchtick Fast Track STTR项目(与威斯康星州大学合作实验室合作)旨在开发和 验证用户友好和高效的基于PIXUL的高通量技术，PIXUL-FFPE，以提取 从FFPE小样本中剪切染色质和DNA，用于表观遗传学分析，用于研究 市场为临床使用铺平了道路。提出了以下目标。在AIM#1(阶段1)中，我们 将设计和验证定制的PIXUL 96孔微孔板，以更有效地提取染色质和DNA 从FFPE组织样本中提取。在Aim#2(阶段2)中，我们将优化PIXUL-FFPE协议以实现快速和高效 从小的FFPE样品中提取染色质，同时最大限度地提高样品完整性。在目标#3(阶段 2)我们将通过对匹配对的比较芯片分析来展示PIXUL-FFPE的翻译效用 新鲜冰冻切片和FFPE胶质母细胞瘤(GBM)切片。在AIM#4(阶段2)中，我们将修改PIXUL仪器 用于从FFPE样品中提取染色质。 由于缺乏可用的数据，表观遗传疗法的搜索速度变慢了。因此，将自定义 改进的PIXUL仪器的96孔板和改进的方案将大大提高FFPE的利用率 标本的研究市场吸引了更多的调查人员去探索浩瀚的 存档的人体组织的储存库，因此推动了精确医学的发展。

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.