smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods.
smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods.
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DOI：
10.1038/s42003-021-01803-0
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发表时间：
2021-03-19
影响因子：
5.9
通讯作者：
Pettini T
中科院分区：
文献类型：
--
作者：
Calvo L;Ronshaugen M;Pettini T
最近，荧光原位杂交技术和成像技术的进步使得能够可视化和计数单个细胞中的单个RNA分子。这大大提高了我们对转录过程的理解。在这里，我们适应最近发表的smiFISH协议（单分子廉价的荧光原位杂交）的整个胚胎在一系列的节肢动物模型物种，也非胚胎组织。使用具有不同光谱的多个荧光团和白色激光共聚焦成像，我们同时检测和分离来自整个胚胎中多达8个不同基因的单个RNA。我们还将联合收割机smiFISH与细胞膜免疫荧光相结合，并提出了一种用于全胚盘胚胎中3D细胞分割和单细胞RNA计数的成像和分析管道。最后，使用全胚胎单细胞RNA计数数据，我们提出了两种替代的单细胞变异性措施，常用的Fano因子，并比较这三种措施的能力，以解决不同方面的单细胞表达变异。在这里，作者将联合收割机单分子廉价FISH（smiFISH）与细胞膜免疫荧光和数学方法相结合，以实现3D图像堆栈中的整个胚胎分割和胚胎每个细胞中多个基因的mRNA定量。
Recently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualization and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability. Here, the authors combine single-molecule inexpensive FISH (smiFISH) with cell membrane immunofluorescence and mathematical methods to enable whole embryo segmentation in 3D image stacks and mRNA quantification of multiple genes in each cell of the embryo.
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影响因子：
21.3
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影响因子：
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作者：
Forrest, KM;Gavis, ER
通讯作者：
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影响因子：
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作者：
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通讯作者：
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作者：
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通讯作者：
Singer, RH
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作者：
FRASCH, M;HOEY, T;LEVINE, M
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