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裹（FFPE）组织档案代表最大的临床注释人 标本存储库。估计数百万可用的FFPE组织样品为该公司提供了广泛的资源 疾病表观遗传途径，生物标志物和药物靶标的发现。然而，这些样品很高 未被充分利用，因为从FFPE样品中提取分子信息的电流方法很慢， 效率低下，劳动密集型，不足以检测基因结合酶（药物靶标），并且具有较低的 吞吐量以充分利用其巨大的研究和临床潜力。对更好的工具有未满足的需求 利用这些存档的临床样品来检索染色质，RNA，DNA和蛋白质 发现个人医学生物标志物。 我们以前已经开发并销售了一个高通量像素超声器，该超声超声音器会产生高强度 96孔微板孔中的聚焦超声（HIFU），以促进染色质，RNA，DNA和蛋白 由培养的细胞和组织制备。 正如我们当前的快速轨道STTR应用程序中提出的那样（现在是第2阶段的第二年），我们已经建立了 使用Pixul从FFPE中提取染色质，DNA和RNA的方案。我们对这些基准测试了 使用微板的FFPE检索方案针对匹配的小鼠冷冻组织（脑，心脏，肾脏和肝脏） 矩阵chip-qpcr/seq，矩阵 - QPCR/seq，矩阵rt-qpcr/seq平台。尽管如此，测试管和热量 块对于分析物的热检索是必需的。如果所有样本准备都可以简化该过程 使用相同的96孔微孔板在一种仪器中完成包括超声处理和加热在内的步骤。因此，一个 当前的STTR赠款的重要目标是设计一种用于处理更通用的像素仪器 FFPE。因此，我们设计了96孔微板双功能实验室像素迭代仪器 这允许对超声和加热/冷却参数进行编程，Pixul H＆C。图书馆准备， 测序套件和Pixul工程成本仍在限制我们优化和商业化Pixul的能力 H＆C用于对包括FFPE的组织的高吞吐量分析。要求行政补充 通过增加NGS测试的组织数量并允许更多 工程资源开发Pixul H＆C商业原型和自定义微型板岩。 全基因组组织多媒体研究为发现提供了宝贵的资源。因此，高 我们建议为NGS优化的吞吐量，快速有效的用户友好双功能Pixul H＆C设备 应用程序和商业化将证明其对研究人员的实用性，以此作为询问的重要手段 FFPE的大量存储库发现疾病途径，候选生物标志物和药物靶标。鉴于 FFPE通常在临床实验室中用于测试分子生物标志物，我们期望Pixul H＆C， 同样的PCR机器也将在临床环境中广泛使用。