mRNA quantification using single-molecule FISH in Drosophila embryos

Abstract

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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Figure 2: Detection of single, smFISH-hybridized mRNAs in Drosophila embryos.
Figure 4: Determining the fluorescence intensity of a single mRNA molecule.
Figure 3: Workflow of the single mRNA molecule detection using Airlocalize.
Figure 5: smFISH enables spatial and quantitative characterization of gene expression in the fly tissue.
Figure 1: Collection and fixing of fly embryos for smFISH.

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Acknowledgements

We thank the NYU Langone Medical Center (NYULMC) Microscopy Laboratory for providing the API DeltaVision personal DV microscope, particularly Y. Deng and M. Cammer for help with the wide-field epifluorescence microscope. We thank V. Schoonderwoert (Scientific Volume Imaging) for his help with the Huygens deconvolution software. This work was supported by the Intramural Research Programs of the US National Institute of Biomedical Imaging and Bioengineering. T.T. is a Howard Hughes Medical Institute (HHMI) fellow of the Jane Coffin Childs Memorial Fund. R.L. is an HHMI investigator. Funding for T.L. was provided by the Howard Hughes Medical Institute.

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T.T. and R.L. implemented quantitative smFISH in Drosophila tissue. H.S. developed iSIM. T.L. developed Airlocalize. All the authors wrote and approved the final version of the manuscript.

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Correspondence to Tatjana Trcek.

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Supplementary information

Supplementary Table 1

Supplementary Table 1. List of sequences of all smFISH probes used in this study. (☼) denotes the position of the fluorophore in the sequence. (PDF 67 kb)

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Trcek, T., Lionnet, T., Shroff, H. et al. mRNA quantification using single-molecule FISH in Drosophila embryos. Nat Protoc 12, 1326–1348 (2017). https://doi.org/10.1038/nprot.2017.030

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