Division of Chemical, Systems & Synthetic Biology, Institute for Infectious Disease & Molecular Medicine, Faculty of Health Sciences, University of Cape Town, 7295 Cape Town, South Africa.
Advanced Medical Bioimaging, Charité - Universitätsmedizin, 10-117 Berlin, Germany.
Cell Rep Methods. 2021 Sep 13;1(5):100068. doi: 10.1016/j.crmeth.2021.100068. eCollection 2021 Sep 27.
Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.
单细胞 RNA 测序技术的进步使得能够根据每个细胞中转录本数量的定量来鉴定细胞亚型。然而，分子（包括 RNA）在空间分布上也可能存在差异。在这里，我们提出了 DypFISH 方法，用于定量研究 RNA 和蛋白质的亚细胞定位。我们引入了一系列分析技术来结合蛋白免疫标记检测单分子 RNA 荧光杂交（smFISH）数据。DypFISH 适用于研究分子聚类模式、mRNA-蛋白质亚细胞定位与微管组织中心取向的关联以及 mRNA-蛋白质空间分布的相互依赖性。我们展示了我们的分析工具如何通过利用细胞微图案来限制细胞结构，从而减少亚细胞 mRNA 分布变化，从而能够对其定位模式进行特征描述，从而获得生物学见解。此外，我们还表明，我们的方法可以应用于生理系统，如骨骼肌纤维。
