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

PIXUL-FFPE：高通量平台，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 - 补充应用。

• 批准号: 10650670
• 负责人: Karol Bomsztyk
• 金额: $ 15.58万
• 依托单位: MATCHSTICK TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-21 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10650670
• 关键词: Administrative Supplement; Antibodies; Archives; Benchmarking; Bioinformatics; Biological Assay; Biological Markers; Biological Specimen Banks; Brain; Cataloging; Catalogs; China; Chromatin; Clinical; Cultured Cells; Custom; DNA; DNA purification; Data; Devices; Diagnosis; Diagnostic; Disease; Disease Pathway; Drug Targeting; Engineering; Enzymes; Epigenetic Process; Europe; Focused Ultrasound; Focused Ultrasound Therapy; Formalin; Freezing; Funding; Genes; Genetic Transcription; Goals; Grant; Heart; Heating; Histopathology; Human; Immunoprecipitation; Kidney; Laboratories; Libraries; Licensing; Link; Liver; Lung; MGMT gene; Medicine; Methods; Methylation; Molecular; Molecular Diagnosis; Molecular Disease; Mus; Muscle; North America; Organ; Paraffin Embedding; Parents; Pathway interactions; Phase; Preparation; Process; Proteins; Proteomics; Protocols documentation; RNA; Reagent; Research; Research Personnel; Resources; Retrieval; Sales; Sampling; Services; Small Business Innovation Research Grant; Small Business Technology Transfer Research; Solvents; Sonication; Specimen; System; Technology; Temperature; Testing; Time; Tissue Banks; Tissue Embedding; Tissue Sample; Tissues; Tube; United States National Institutes of Health; Universities; Washington; Work; base; biomarker discovery; candidate marker; chromatin immunoprecipitation; clinical practice; cost; design; epigenome; genome-wide; high throughput analysis; human tissue; improved; instrument; molecular marker; multiple omics; next generation sequencing; programs; prototype; repository; research and development; sample fixation; tissue archive; tissue processing; tool; transcriptome; transcriptome sequencing; ultrasound; 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 a 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, inefficient, 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, RNA, DNA and proteins for the discovery of biomarkers for personal medicine. We have previously developed and marketed a high-throughput PIXUL sonicator which generates high-intensity focused ultrasound (HIFU) in wells of 96-well microplate to advance chromatin, RNA, DNA and protein preparation from cultured cells and tissues. As proposed in our current Fast Track STTR application (now in year 2 of Phase 2), we have established protocols for extraction of chromatin, DNA, and RNA from FFPEs using PIXUL. We have benchmarked these FFPE retrieval protocols against matched mouse frozen tissues (brain, heart, kidney and liver) using microplate Matrix-ChIP-qPCR/seq, Matrix-MeDIP-qPCR/seq, Matrix-RT-qPCR/seq platforms. Still, test-tubes and heat block are necessary for heat retrieval of analytes. The process can be simplified if all the sample preparation steps, including sonication and heating, are done in one instrument using the same 96-well microplate. Thus, an important goal of the current STTR grant was to engineer a more versatile PIXUL instrument for processing FFPEs. Accordingly, we designed a 96-well microplate dual functionality laboratory PIXUL iteration instrument that allows to program ultrasound and heating/cooling parameters, PIXUL H&C. The library preparation, sequencing kits and PIXUL engineering costs are still limiting our ability to optimize and commercialize PIXUL H&C for high throughput analysis of tissues including FFPEs. The administrative supplement is requested to expedite PIXUL-FFPE R&D by increasing the number of tissues for NGS testing and allowing more engineering resources to develop PIXUL H&C commercial prototype and custom microplates. Genome-wide tissue multiomic studies have provided valuable resources for discoveries. Thus, the high- throughput, fast and efficient user-friendly dual-function PIXUL H&C device that we propose to optimize for NGS applications and to commercialize will demonstrate its utility to researchers as an important means to interrogate the vast repositories of FFPEs to discover disease pathways, candidate biomarkers and drug targets. Given that FFPE are most often used in clinical laboratories for testing molecular biomarkers we expect that PIXUL H&C, alike PCR machines, will also have wide use in clinical settings.

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点的发现。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缓慢，因此未得到充分利用， 效率低下、劳动密集型、检测基因结合酶（药物靶点）不够灵敏且检测率低 吞吐量以充分发挥其巨大的研究和临床潜力。对更好工具的需求尚未得到满足 利用这些存档的临床样本检索染色质、RNA、DNA 和蛋白质，用于 个人医学生物标志物的发现。 我们之前开发并销售了一款高通量 PIXUL 超声波发生器，可产生高强度 在 96 孔微孔板的孔中进行聚焦超声 (HIFU) 以促进染色质、RNA、DNA 和蛋白质 从培养的细胞和组织中制备。 正如我们当前的快速通道 STTR 申请（现在处于第二阶段的第二年）中所提议的，我们已经建立了 使用 PIXUL 从 FFPE 中提取染色质、DNA 和 RNA 的方案。我们对这些进行了基准测试 使用微孔板针对匹配的小鼠冷冻组织（大脑、心脏、肾脏和肝脏）进行 FFPE 检索方案 Matrix-ChIP-qPCR/seq、Matrix-MeDIP-qPCR/seq、Matrix-RT-qPCR/seq 平台。尽管如此，试管和加热 块对于分析物的热回收是必要的。如果所有的样品制备过程都可以简化 包括超声处理和加热在内的步骤均使用相同的 96 孔微孔板在一台仪器中完成。因此，一个 当前 STTR 拨款的重要目标是设计一种更通用的 PIXUL 仪器进行处理 FFPE。据此，我们设计了96孔微孔板双功能实验室PIXUL迭代仪 允许对超声波和加热/冷却参数进行编程，PIXUL H&C。文库准备， 测序试剂盒和 PIXUL 工程成本仍然限制我们优化和商业化 PIXUL 的能力 H&C 用于组织（包括 FFPE）的高通量分析。请求行政补充 通过增加用于 NGS 测试的组织数量并允许更多组织来加快 PIXUL-FFPE 研发 开发 PIXUL H&C 商业原型和定制微孔板的工程资源。 全基因组组织多组学研究为发现提供了宝贵的资源。因此，高 高通量、快速、高效、用户友好的双功能 PIXUL H&C 设备，我们建议针对 NGS 进行优化 应用和商业化将向研究人员展示其作为询问的重要手段的实用性 巨大的 FFPE 存储库，用于发现疾病途径、候选生物标志物和药物靶标。鉴于 FFPE 最常用于临床实验室测试分子生物标志物，我们预计 PIXUL H&C、 与 PCR 机器一样，也将在临床环境中广泛使用。