xMD-miRNA-Seq estimates cell-specific miRNA expression

项目摘要/摘要 在过去的几年里，人们已经重要地认识到，为了真正理解 人类疾病我们不仅要了解患病的组织，还要了解这些组织中的细胞。到那时候 最后，我们需要工具来捕捉这些细胞并探索它们的表达模式。我们已经开发出一种 名为xMD-miRNA-Seq的技术就是为了做到这一点。XMD代表表达微分离，它 允许我们一次从任何固定的组织来源收集一种类型的细胞。我们已经开发出一种方法来使用 对这些细胞进行小RNA测序，以捕捉这些细胞中microRNAs的表达谱。 MicroRNAs(MiRNAs)是参与疾病和发育的小的、调节的RNAs。虽然这是一个 这项令人兴奋的强大技术旨在帮助理解细胞表达，但它的效率仍然相对较低。 因此，我们提出了一套优化方案，使该技术具有广泛的适应性。 我们已经提出了优化细胞从载玻片移动到贴膜的捕获的方法 调节功能包括温度、光照强度、背景类型甚至膜 它本身。我们还试图通过xMD过程的步骤来限制RNA的损失，方法是对某些 含有用于去除核糖核酸酶的化学物质的试剂。最后，我们将与现有的和新的 方法最大限度地提高在小RNA测序运行中读取的miRNAs的百分比。最终，这些 修改将大大增强xMD-miRNA-seq方法的价值，并将使我们能够获得 几乎在任何疾病环境中几乎任何人类细胞类型的miRNA图谱。这对癌症是很重要的。 生物学、免疫学、发育、心血管疾病、神经疾病和许多其他环境。

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.