xMD-miRNA-Seq estimates cell-specific miRNA expression

xMD-miRNA-Seq 估计细胞特异性 miRNA 表达

• 批准号: 10240467
• 负责人: MARC K HALUSHKA
• 金额: $ 31.11万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10240467
• 关键词: Adoption; Antigens; Archives; Atlases; Basic Science; Biological Markers; Biology; Cancer Biology; Cardiovascular Diseases; Cardiovascular system; Cells; Chemicals; Collection; Colon; Color; Complement; Cultured Cells; Data; Development; Disease; Doctor of Philosophy; Endothelial Cells; Epithelial; Epithelial Cells; Ethylenes; Fibroblasts; Formalin; Generations; Genes; Glass; Glomerular capsule structure; Goals; Health; High temperature of physical object; Human; Human Resources; Immunohistochemistry; Immunology; Kidney; Knowledge; Lead; Lifting; Light; Membrane; Methodology; Methods; MicroRNAs; Microdissection; Modification; Normal tissue morphology; Paraffin Embedding; Pathologist; Phenotype; Pigments; Preparation; Process; Protocols documentation; RNA; RNA Folding; RNA library; Reagent; Research; Research Personnel; Resources; Retrieval; Ribonucleases; Ribosomal RNA; Ribosomes; Role; Running; Sequence Alignment; Slide; Small RNA; Source; Specimen; Surface; Surgical Pathology; Techniques; Technology; Temperature; Therapeutic; Time; Tissue Embedding; Tissues; Work; animal tissue; base; cell type; cost effective; design; glomerular endothelium; high throughput technology; human disease; human tissue; improved; in vivo; inhibitor/antagonist; interest; laser capture microdissection; light intensity; melting; mesangial cell; miRNA expression profiling; nervous system disorder; new technology; podocyte; research study; success; technology development; tool; transcriptome sequencing; vinyl acetate

Project Summary/Abstract There has been an important realization over the past several years that in order to truly understand human disease we must understand not just the diseased tissues, but rather the cells in those tissues. To that end, we need tools that can capture those cells and explore their expression profiles. We have developed a technology called xMD-miRNA-Seq to do just that. The xMD stands for expression microdissection and it allows us to collect one cell type at a time from any fixed tissue source. We have developed a way to use those cells for small RNA sequencing to capture the expression profile of microRNAs from just these cells. microRNAs (miRNAs) are small, regulatory RNAs involved in disease and development. Although this is an exciting, powerful technique designed to help understand cell expression, it remains relatively inefficient. Therefore, we have proposed a set of optimizations to make the technique widely adaptable. We have proposed to optimize the capture of cells moving from a slide to an affixed membrane by adjusting features including the temperature, the light intensity, the background type and even the membrane itself. We also seek to limit the loss of RNA through the steps of the xMD process by pretreating certain reagents with chemicals designed to remove RNAses. Finally, we will work with both established and new methods to maximize the percent of miRNAs that are read in a small RNA sequencing run. Ultimately, these modifications will greatly enhance the value of the xMD-miRNA-seq method and will allow us to obtain the miRNA profile of almost any human cell type in almost any disease setting. This will be important for cancer biology, immunology, development, cardiovascular disease, neurologic disorders and many other settings.

项目总结/摘要 在过去的几年里，有一个重要的认识，为了真正了解 我们不仅要了解患病的组织，还要了解这些组织中的细胞。与 最后，我们需要能够捕获这些细胞并探索其表达谱的工具。我们已经开发出一种 一种叫做xMD-miRNA-Seq的技术来做到这一点。xMD代表表达显微切割， 允许我们从任何固定的组织来源一次收集一种细胞类型。我们发明了一种方法 这些细胞进行小RNA测序，以捕获来自这些细胞的microRNA的表达谱。 microRNA（miRNAs）是一种小的调控RNA，参与疾病和发育。虽然这是一个 虽然这项技术是一项令人兴奋的、强大的技术，旨在帮助理解细胞表达，但它仍然相对低效。 因此，我们提出了一组优化，使该技术具有广泛的适应性。 我们已经提出通过以下方式来优化从载玻片移动到固定膜的细胞的捕获： 调节功能，包括温度，光强度，背景类型，甚至膜 本身我们还试图通过预处理某些RNA来限制通过xMD过程步骤的RNA损失。 含有去除核糖核酸酶的化学试剂。最后，我们将与既有和新的 最大化在小RNA测序运行中读取的miRNA百分比的方法。最终，这些 修改将大大提高xMD-miRNA-seq方法的价值，并使我们能够获得 几乎任何疾病背景下几乎任何人类细胞类型的miRNA谱。这对癌症很重要 生物学、免疫学、发育学、心血管疾病、神经系统疾病和许多其他环境。