SCAMPR, a single-cell automated multiplex pipeline for RNA quantification and spatial mapping.
SCAMPR, a single-cell automated multiplex pipeline for RNA quantification and spatial mapping.
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DOI：
10.1016/j.crmeth.2022.100316
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发表时间：
2022-10-24
期刊：
影响因子：
--
通讯作者：
Levitt P
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文献类型：
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作者：
Ali Marandi Ghoddousi R;Magalong VM;Kamitakahara AK;Levitt P
空间基因表达，通常通过原位杂交实现，是神经系统细胞类型的地形表型的基本工具。新开发的技术允许在单细胞分辨率下可视化多个mrna，并极大地扩展了将基因表达与组织地形联系起来的能力，但在这些高维数据集的有效量化和分析方面存在挑战。因此，我们开发了单细胞RNA自动化多路管道（SCAMPR），使用双重免疫组织化学- rnascope协议促进神经元细胞体的快速准确分割，并使用开源图像处理和自动分割工具对低丰度和高丰度mRNA信号进行量化。使用SCAMPR的原理证明主要集中在外周（迷走神经结节）和中央（视觉皮质）神经元基因表达的空间定位上。通过鉴定早期生活应激对迷走神经神经元亚型基因表达的影响，证明了SCAMPR的分析有效性。双HiPlex-IHC允许识别PNS和CNS中的神经元边界，组间基因表达差异通过ImageJ/R编码识别。SCAMPR快速，易于使用，成本效益高，神经元中空间mRNA表达的定量分析在准确性，计算和时间密集方面存在挑战。现有的依靠核标记（DAPI）来区分相邻细胞的方法缺乏检测细胞质中mRNA表达的精度。此外，依赖点计数的定量方法可能产生大量可变的数据集，这可能会低估高表达mrna的数量。为了克服这些方法学上的障碍，我们开发了SCAMPR管道，可以快速、准确地分割神经元细胞体边界，绘制基因表达图谱，并对组织切片中的mRNA表达进行高维量化和分析。smFISH方法的广泛使用将大大受益于易于获得的量化和分析工具。Ali Marandi Ghoddousi等人提出了SCAMPR，这是一个易于使用的开源管道，用于在全细胞水平上准确量化smFISH信号。SCAMPR有助于拓扑基因表达分析和确定实验组之间的基因表达差异。
Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes. SCAMPR is a pipeline for quantification and spatial analysis of gene expression Dual HiPlex-IHC allows for identification of neuronal boundaries in the PNS and CNS Gene expression differences between groups are identified by ImageJ/R code SCAMPR is rapid, easy to use, and cost effective Quantitative analysis of spatial mRNA expression in neurons presents challenges in terms of accuracy and being computationally and time intensive. Existing methods that rely on nuclear labeling (DAPI) to distinguish adjoining cells lack the precision to detect mRNA expression in the cytoplasm. In addition, quantification methods that rely on puncta counts can generate large, variable datasets that potentially undercount highly expressed mRNAs. To overcome these methodological barriers, we developed the SCAMPR pipeline that allows for fast, accurate segmentation of neuronal cell body boundaries, topographic gene expression mapping, and high-dimensional quantification and analysis of mRNA expression in tissue sections. The widespread use of smFISH methods will benefit greatly from accessible quantification and analysis tools. Ali Marandi Ghoddousi et al. present SCAMPR, an easy-to-use, open-source pipeline for accurately quantifying smFISH signal at a whole-cell level. SCAMPR facilitates topological gene expression analyses and identification of gene expression differences between experimental groups.