原位杂交
生物
共焦
共焦显微镜
细胞生物学
分子生物学
寡核苷酸
信使核糖核酸
荧光原位杂交
基因表达
基因
遗传学
几何学
染色体
数学
作者
Tatjana Trcek,Timothée Lionnet,Hari Shroff,Ruth Lehmann
出处
期刊:Nature Protocols
[Springer Nature]
日期:2016-06-08
卷期号:12 (7): 1326-1348
被引量:81
标识
DOI:10.1038/nprot.2017.030
摘要
Single-molecule fluorescence in situ hybridization (smFISH) enables mRNA quantification while preserving spatial information. Trcek et al. describe an smFISH procedure for Drosophila embryos, using wide-field, confocal and structured illumination miscroscopy. Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured illumination microscopy (iSIM, a super-resolution imaging approach) and a spot-detection algorithm. Fixed Drosophila embryos are hybridized in solution with a mixture of smFISH probes, mounted onto coverslips and imaged in 3D. Individual fluorescently labeled mRNAs are then localized within tissues and counted using spot-detection software to generate quantitative, spatially resolved gene expression data sets. With minimum guidance, a graduate student can successfully implement this protocol. The smFISH procedure described here can be completed in 4–5 d.
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