Droplet Microfluidic Platform for Ultralow Input Epigenetics

用于超低输入表观遗传学的液滴微流控平台

• 批准号: 9015419
• 负责人: Ryan C Bailey
• 金额: $ 11.05万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-16 至 2016-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9015419
• 关键词: Address; Aging; Antibodies; Automation; Basic Science; Benchmarking; Biological Assay; Biopsy; Cancer Biology; Cell Nucleus; Cells; Chromatin; Clinic; Clinical; Complex; Cytolysis; DNA; DNA Modification Process; DNA Repair; DNA Sequence; DNA analysis; DNA biosynthesis; DNA purification; DNA-Protein Interaction; Development; Device Designs; Devices; Digestion; Dinucleosome; Encapsulated; Epigenetic Process; Genetic Transcription; Genome; Genomics; Goals; Gold; Health; Histones; Liquid substance; Malignant Neoplasms; Methods; Microfluidic Microchips; Microfluidics; Molecular Target; Mono-S; Neoplasm Circulating Cells; Nucleosomes; Oils; Phase; Play; Population; Post-Translational Protein Processing; Preparation; Process; Proteins; Proteomics; Protocols documentation; Publishing; Recovery; Reporting; Research; Resolution; Role; Sample Size; Sampling; Side; Signal Transduction; Stem cells; Technology; Testing; Time; Touch sensation; Translational Research; Translations; Untranslated RNA; Validation; Variant; Writing; aqueous; base; cancer diagnosis; cancer stem cell; cancer therapy; carcinogenesis; cell fate specification; chromatin immunoprecipitation; cost effective; epigenomics; genome-wide; histone modification; individualized medicine; insight; magnetic beads; nanolitre scale; non-histone protein; operation; parallelization; personalized cancer therapy; personalized strategies; point of care; protein complex; skills; sound; synthetic construct; technology development; tool; transcription factor; treatment strategy; tumor; tumor heterogeneity

 DESCRIPTION (provided by applicant): Epigenomic analyses are playing increasingly prominent roles in the development of personalized strategies for treating cancer. However, the translation of fundamental epigenomic insight to the clinic is wrought with challenges. Take for example the study of post-translational histone protein modifications, which can serve to either promote or repress the transcription of pendant DNA sequences. Analyses of these critically important interactions bridge genomics and proteomics and present significant challenges in the clinical setting. Chromatin immunoprecipitation (ChIP) is the method of choice for analyzing protein-DNA interactions and the basic method involves fragmentation of chromatin, separation of modified proteins using antibodies and magnetic beads, and subsequent analysis of the associated DNA by qPCR or sequencing. While sounding simple, a typical ChIP workflow involves ~30 steps, takes 4+ days, and requires 106-107 cells as input. These requirements significantly limit the applicability of ChIP in a clinical setting-particularly when minimal sampl is available, such as in the analysis of tumor biopsies, stem cells, or circulating tumor cells. Microfluidic devices offer many attractive benefits over traditional macro-scale methods including reduced volume requirements, parallelization capability, and automated operation, which make them particularly well- suited to sample-constrained epigenetic analyses. A handful of recent reports suggest a substantial opportunity for microfluidically-enabled ChIP analyses; however, there is still considerable room for further improvement. We propose to develop a powerful and versatile, droplet microfluidics-based, nanoliter-scale Chromatin ImmunoCapture (nChIC) platform suitable for individualized medicine applications. Droplet microfluidics offer several benefits, including rapid, controlled, and efficient fluid handling, and the capacity to handle variable sample sizes, since devices can accommodate larger samples by simply operating for longer periods of time. Our nChIC platform will incorporate every major step in the ChIP workflow into an automated device, including cell lysis, chromatin digestion, immunocapture, and DNA purification. Importantly, these processes will be carried out at the single cell level, which promises to provide unique insights into epigenomic tumor heterogeneity. Beyond single cells, the unprecedented ability to handle samples of variable input will also facilitate robust validation against traditional ChIP assays to demonstrate broad genomic coverage. Taken together, we feel that the nChIC platform will be a powerful new tool that enables the translation of epigenomic insight into individualized cancer treatment at the point of care.

 描述(由申请人提供)：表观基因组分析在个性化癌症治疗策略的发展中发挥着越来越重要的作用。然而，将基本的表观基因组学洞察转化到临床是充满挑战的。以翻译后组蛋白蛋白修饰的研究为例，它可以促进或抑制悬挂DNA序列的转录。对这些至关重要的相互作用的分析在基因组学和蛋白质组学之间架起了桥梁，并在临床环境中提出了重大挑战。染色质免疫沉淀(ChIP)是分析蛋白质-DNA相互作用的首选方法，基本方法包括染色质碎裂、使用抗体和磁珠分离修饰的蛋白质，以及随后通过定量聚合酶链式反应或测序分析相关DNA。虽然听起来很简单，但一个典型的芯片工作流程需要大约30个步骤，需要4天以上的时间，需要106-107个细胞作为输入。这些要求大大限制了芯片在临床环境中的适用性--特别是当有最小样本可用时，例如在肿瘤活检、干细胞或循环肿瘤细胞的分析中。与传统的宏观方法相比，微流控设备具有许多吸引人的优点，包括减少体积需求、并行化能力和自动化操作，这使得它们特别适合于样本受限的表观遗传学分析。最近的几份报告表明，微流控芯片分析有很大的机会；然而，仍有相当大的改进空间。我们建议开发一种功能强大且通用的、基于液滴微流体的、纳升规模的染色质免疫捕获(NChIC)平台，适用于个性化药物应用。液滴微流体具有几个优点，包括快速、可控和高效的流体处理，以及处理不同样品大小的能力，因为设备只需更长时间的操作就可以容纳更大的样品。我们的nChIC平台将把芯片工作流程中的每个主要步骤整合到一个自动化设备中，包括细胞裂解、染色质消化、免疫捕获和DNA纯化。重要的是，这些过程将在单细胞水平上进行，这有望为表观基因组肿瘤的异质性提供独特的见解。除了单个细胞，前所未有的处理可变输入样本的能力也将促进相对于传统芯片分析的强大验证，以证明广泛的基因组覆盖。综上所述，我们认为nChIC平台将是一个强大的新工具，能够在护理点将表观基因组学见解转化为个性化的癌症治疗。