Quantifying tissue-specific overexpression of FOXO in Drosophila via mRNA fluorescence in situ hybridization using branched DNA probe technology

Anna C. Blice-Baum, Georg Vogler, Meera C. Viswanathan, Bosco Trinh, Worawan B. Limpitikul, Anthony Cammarato

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages171-190
Number of pages20
DOIs
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
Volume1890
ISSN (Print)1064-3745

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Fingerprint Dive into the research topics of 'Quantifying tissue-specific overexpression of FOXO in Drosophila via mRNA fluorescence in situ hybridization using branched DNA probe technology'. Together they form a unique fingerprint.

Cite this