Almansour Faisal, Keikhosravi Adib, Pegoraro Gianluca, Misteli Tom
National Cancer Institute, National Institute of Health.
Res Sq. 2024 Feb 22:rs.3.rs-3970096. doi: 10.21203/rs.3.rs-3970096/v1.
The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are non-random, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection. These optimized DNA and RNA FISH protocols, implemented in a 384-well plate format alongside automated image and data analysis, enable accurate detection of chromatin loci and their gene expression status across a large cell population with allele-level resolution. We successfully visualized and DNA and RNA in multiple cell types, and we determined the radial position of active and inactive and alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.
基因组在细胞核内的空间排列是细胞组织和功能的关键方面，对基因表达和调控具有重要意义。虽然所有基因组组织特征，如环、结构域和径向定位，都是非随机的，但它们具有高度的单细胞变异性。成像方法非常适合可视化、测量和研究基因组组织中的单细胞异质性。在这里，我们描述了两种通过高通量荧光原位杂交（FISH）检测单个基因等位基因的DNA和RNA的方法。我们优化了组合DNA/RNA FISH方法，采用同时或顺序检测。这些优化的DNA和RNA FISH方案以384孔板形式实施，并结合自动图像和数据分析，能够在等位基因水平分辨率下，准确检测大量细胞群体中的染色质位点及其基因表达状态。我们成功地在多种细胞类型中可视化了DNA和RNA，并确定了活跃和不活跃等位基因的径向位置。这些优化的DNA/RNA检测方法是用于在单等位基因水平和高通量下绘制染色质特征和基因活性的通用且灵敏的工具。
