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Quantifying Tissue-Specific Overexpression of FOXO in Drosophila via mRNA Fluorescence In Situ Hybridization Using Branched DNA Probe Technology

  • Anna C. Blice-BaumEmail author
  • Georg VoglerEmail author
  • Meera C. Viswanathan
  • Bosco Trinh
  • Worawan B. Limpitikul
  • Anthony Cammarato
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1890)

Abstract

While the highly conserved FOXO transcription factors have been studied in Drosophila melanogaster for decades, the ability to accurately control and measure their tissue-specific expression is often cumbersome due to a lack of reagents and to limited, nonhomogeneous samples. The need for quantitation within a distinct cell type is particularly important because transcription factors must be expressed in specific amounts to perform their functions properly. However, the inherent heterogeneity of many samples can make evaluating cell-specific FOXO and/or FOXO load difficult. Here, we describe an extremely sensitive fluorescence in situ hybridization (FISH) approach for visualizing and quantifying multiple mRNAs with single-cell resolution in adult Drosophila cardiomyocytes. The procedure relies upon branched DNA technology, which allows several fluorescent molecules to label an individual transcript, drastically increasing the signal-to-noise ratio compared to other FISH assays. This protocol can be modified for use in various small animal models, tissue types, and for assorted nucleic acids.

Key words

Fluorescence in situ hybridization FISH Drosophila melanogaster Heart tube Dorsal vessel Branched DNA bDNA ViewRNA RNAscope 

Notes

Acknowledgments

We thank Holly Howarth for excellent technical assistance. This work was supported by R01HL124091 (to A.C.) and Cabrini University Academic Affairs funds (to A.C.B.). For questions, please contact A.C.B. (anna.c.blice-baum@cabrini.edu) or G.V. (gvogler@sbpdiscovery.org).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Anna C. Blice-Baum
    • 1
    • 2
    Email author
  • Georg Vogler
    • 3
    Email author
  • Meera C. Viswanathan
    • 1
  • Bosco Trinh
    • 3
  • Worawan B. Limpitikul
    • 4
  • Anthony Cammarato
    • 1
    • 5
  1. 1.Division of Cardiology, Department of MedicineJohns Hopkins University School of MedicineBaltimoreUSA
  2. 2.Science Department, Iadarola Center for Science, Education and TechnologyCabrini UniversityRadnorUSA
  3. 3.Development, Aging and Regeneration ProgramSanford Burnham Prebys Medical Discovery InstituteLa JollaUSA
  4. 4.Department of Biomedical EngineeringJohns Hopkins University School of MedicineBaltimoreUSA
  5. 5.Department of PhysiologyJohns Hopkins University School of MedicineBaltimoreUSA

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