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Related Experiment Video

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mRNA quantification using single-molecule FISH in Drosophila embryos.

Tatjana Trcek1, Timothée Lionnet2, Hari Shroff3

  • 1Howard Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU School of Medicine, New York, New York, USA.

Nature Protocols
|June 9, 2017
PubMed
Summary
This summary is machine-generated.

This study presents a single-molecule fluorescence in situ hybridization (smFISH) protocol for precisely quantifying mRNA within Drosophila embryos. This method preserves spatial information lost in other techniques, enabling detailed gene expression analysis.

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Area of Science:

  • Developmental Biology
  • Molecular Biology
  • Genetics

Background:

  • Gene expression regulation is crucial for development and tissue function.
  • Traditional methods like PCR and sequencing often lose spatial mRNA information.
  • Single-molecule fluorescence in situ hybridization (smFISH) retains cellular mRNA spatial data.

Purpose of the Study:

  • To describe a robust smFISH protocol for quantifying single mRNAs in Drosophila embryos.
  • To enable spatially resolved gene expression analysis at the subcellular level.
  • To provide a method that complements existing transcriptomic techniques.

Main Methods:

  • Utilized commercially available smFISH probes (fluorescently labeled DNA oligonucleotides).
  • Employed wide-field epifluorescence, confocal, or instant structured illumination microscopy (iSIM).
  • Applied a spot-detection algorithm for mRNA localization and counting in 3D.

Main Results:

  • Successfully quantified single mRNAs in fixed Drosophila embryos with subcellular resolution.
  • Generated quantitative, spatially resolved gene expression datasets.
  • Demonstrated the protocol's feasibility for graduate students, completable in 4-5 days.

Conclusions:

  • The described smFISH protocol effectively preserves and quantifies spatial mRNA information in Drosophila embryos.
  • This technique offers a valuable tool for studying gene expression dynamics in developmental contexts.
  • The protocol is accessible and efficient for generating high-resolution transcriptomic data.