Using single-molecule fluorescence in situ hybridization and immunohistochemistry to count RNA molecules in single cells in zebrafish embryos.
Using single-molecule fluorescence in situ hybridization and immunohistochemistry to count RNA molecules in single cells in zebrafish embryos.
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DOI：
10.1016/j.xpro.2022.102020
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发表时间：
2023-03-17
期刊：
影响因子：
--
通讯作者：
Ozbudak, Ertugrul M.
中科院分区：
文献类型：
--
作者：
Keseroglu, Kemal;Zinani, Oriana Q. H.;Ozbudak, Ertugrul M.
驯服基因表达变异性对于胚胎发育期间的稳健模式形成至关重要。在这里，我们描述了一个优化的协议，单分子荧光原位杂交和免疫组织化学在斑马鱼胚胎。我们详细介绍了如何计算分割时钟RNA和计算它们在相邻细胞之间的变异性。这种方法很容易适用于计算任何基因的RNA数量，并计算不同生物环境中相邻细胞之间的转录变异性。有关本方案使用和执行的完整详细信息，请参见凯斯金等人。（2018），Zinani et al.（2021），和Zinani et al.（2022年）。斑马鱼胚胎中smFISH结合免疫组织化学的优化方案单转录物共聚焦成像的样品制备定量给定组织中单细胞中的RNA分子定量相邻细胞中的转录变异性出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。驯服基因表达变异性对于胚胎发育期间的稳健模式形成至关重要。在这里，我们描述了一个优化的协议，单分子荧光原位杂交和免疫组织化学在斑马鱼胚胎。我们详细介绍了如何计算分割时钟RNA和计算它们在相邻细胞之间的变异性。这种方法很容易适用于计算任何基因的RNA数量，并计算不同生物环境中相邻细胞之间的转录变异性。
Taming gene expression variability is critical for robust pattern formation during embryonic development. Here, we describe an optimized protocol for single-molecule fluorescence in situ hybridization and immunohistochemistry in zebrafish embryos. We detail how to count segmentation clock RNAs and calculate their variability among neighboring cells. This approach is easily adaptable to count RNA numbers of any gene and calculate transcriptional variability among neighboring cells in diverse biological settings. For complete details on the use and execution of this protocol, please refer to Keskin et al. (2018), Zinani et al. (2021), and Zinani et al. (2022). Optimized protocol for smFISH combined with immunohistochemistry in zebrafish embryos Sample preparation for confocal imaging of single transcripts Quantifying RNA molecules in single cells in a given tissue Quantifying transcriptional variability among neighboring cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Taming gene expression variability is critical for robust pattern formation during embryonic development. Here, we describe an optimized protocol for single-molecule fluorescence in situ hybridization and immunohistochemistry in zebrafish embryos. We detail how to count segmentation clock RNAs and calculate their variability among neighboring cells. This approach is easily adaptable to count RNA numbers of any gene and calculate transcriptional variability among neighboring cells in diverse biological settings.
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影响因子：
8.8
作者：
Keskin S;Devakanmalai GS;Kwon SB;Vu HT;Hong Q;Lee YY;Soltani M;Singh A;Ay A;Özbudak EM
通讯作者：
Özbudak EM
影响因子：
5.4
作者：
Gross-Thebing T;Paksa A;Raz E
通讯作者：
Raz E
影响因子：
5.8
作者：
Zinani, Oriana Q. H.;Keseroglu, Kemal;Dey, Supravat;Ay, Ahmet;Singh, Abhyudai;Ozbudak, Ertugrul M.
通讯作者：
Ozbudak, Ertugrul M.
影响因子：
64.8
作者：
Zinani OQH;Keseroğlu K;Ay A;Özbudak EM
通讯作者：
Özbudak EM