Rieger Aja M, Havixbeck Jeffrey J, Barreda Daniel R
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada.
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada; Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada.
J Immunol Methods. 2015 Aug;423:111-9. doi: 10.1016/j.jim.2015.04.021. Epub 2015 May 19.
Fluorescent in situ hybridization (FISH) is a powerful technique for the detection of RNA or DNA within cells and tissues, which provides a unique link between molecular and cell biology. This technique is broadly applicable across a range of biological systems. While FISH has been previously adapted to flow-based platforms, their use remains limited because of procedural challenges and costs associated with commercial kits. Herein we present a protocol that modifies existing techniques to sensitively and specifically detect and examine RNA expression patterns in primary cells and cell lines using flow cytometry (expression-FISH; X-FISH). As relevant examples, we show how this technique can be used to monitor changes in mRNA expression following activation, how it can be combined with antibody staining to study RNA and protein in the same sample, and how it can help distinguish among subsets in a mixed cell population. X-FISH can integrate multiple probes and can be performed in conjunction with other assays, allowing for informative multiparametric analyses and increased statistical robustness. For non-classical comparative animal models this procedure provides a time saving alternative to de novo production of antibody-based markers. Finally, X-FISH provides an economical solution that is applicable to conventional as well as multi-spectral imaging flow cytometry platforms.
荧光原位杂交（FISH）是一种用于检测细胞和组织内RNA或DNA的强大技术，它在分子生物学和细胞生物学之间建立了独特的联系。该技术广泛适用于一系列生物系统。虽然FISH此前已被应用于基于流式细胞仪的平台，但其应用仍然有限，原因在于操作上的挑战以及与商业试剂盒相关的成本。在此，我们提出一种方案，该方案通过改进现有技术，利用流式细胞术灵敏且特异地检测和研究原代细胞及细胞系中的RNA表达模式（表达FISH；X-FISH）。作为相关示例，我们展示了该技术如何用于监测激活后mRNA表达的变化，如何与抗体染色相结合以在同一样本中研究RNA和蛋白质，以及如何帮助区分混合细胞群体中的亚群。X-FISH可以整合多个探针，并可与其他检测方法联合进行，从而实现信息丰富的多参数分析并增强统计稳健性。对于非经典的比较动物模型，该方法为从头生产基于抗体的标志物提供了一种节省时间的替代方案。最后，X-FISH提供了一种经济的解决方案，适用于传统以及多光谱成像流式细胞仪平台。
