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

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、生物标志物和药物靶点的发现。然而，这些样本是高度 未得到充分利用是因为目前从FFPE样本中提取分子信息的方法很慢， 效率低、劳动密集型、检测基因结合酶(药物靶标)不够灵敏，且 生产能力，以充分发挥其巨大的研究和临床潜力。对更好的工具的需求还没有得到满足 利用这些存档的临床样本提取染色质、RNA、DNA和蛋白质 个人药物生物标志物的发现。 我们之前已经开发并销售了一种高通量的PIXUL超声仪，它可以产生高强度的 聚焦超声(HIFU)在96孔微孔板中促进染色质、RNA、DNA和蛋白质 从培养的细胞和组织中制备。 正如我们目前的Fast Track STTR应用程序(现已进入第二阶段的第二年)所建议的那样，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 利用微孔板对匹配的小鼠冷冻组织(脑、心、肾和肝)进行FFPE检索 MATRIX-CHIP-qPCR/seq、Matrix-MeDIP-qPCR/seq、Matrix-RT-qPCR/seq平台。不过，试管和热能 对于分析物的热回收来说，块是必要的。如果所有样品的制备都可以简化过程 步骤，包括超声波和加热，在一个仪器中使用相同的96孔微孔板完成。因此，一个 目前STTR赠款的重要目标是设计一种更多功能的PIXUL处理仪器 FFPE。据此，我们设计了一台96孔微板双功能实验室PIXUL迭代仪器 它允许对超声波和加热/冷却参数进行编程，PIXUL H&C。 测序试剂盒和PIXUL工程成本仍然限制着我们优化PIXUL并将其商业化的能力 用于包括FFPE在内的组织的高通量分析。请提供行政副刊以 通过增加用于NGS测试的组织数量并允许 开发PIXUL H&C商用原型和定制微型板的工程资源。 全基因组的组织多组学研究为发现提供了宝贵的资源。因此，最高- 吞吐量、快速高效的用户友好型双功能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审问的重要手段的实用性 用于发现疾病途径、候选生物标记物和药物靶点的大量FFPE存储库。考虑到 FFPE最常用于临床实验室测试分子生物标记物，我们预计PIXUL H&C， 与PCR机一样，PCR机也将在临床环境中广泛使用。

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.