Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA 02215, USA.
STAR Protoc. 2023 Sep 15;4(3):102463. doi: 10.1016/j.xpro.2023.102463. Epub 2023 Jul 22.
FISH-Flow (fluorescence in situ hybridization-flow cytometry) involves hybridizing fluorescent oligos to RNA and quantifying fluorescence at a single-cell level using flow cytometry. Here, we present a FISH-Flow protocol to quantify nascent 47S and mature 18S and 28S rRNAs in mouse and human cells, including rRNA quantification across cell cycle stages using DNA staining. We describe steps for cell preparation, hybridization of fluorescent probes against rRNA, and DNA staining. We then detail procedures for flow cytometry and data analysis. For complete details on the use and execution of this protocol, please refer to Antony et al. (2022)..
荧光原位杂交-流式细胞术（FISH-Flow）涉及将荧光寡核苷酸与 RNA 杂交，并使用流式细胞术在单细胞水平上定量荧光。在这里，我们提供了一种 FISH-Flow 方案，用于定量小鼠和人类细胞中的新生 47S 和成熟 18S 和 28S rRNA，包括使用 DNA 染色对细胞周期各阶段的 rRNA 进行定量。我们描述了细胞准备、针对 rRNA 的荧光探针杂交以及 DNA 染色的步骤。然后，我们详细介绍了流式细胞术和数据分析的程序。有关该方案使用和执行的完整详细信息，请参阅 Antony 等人（2022 年）。
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