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 Ross 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.

LanguageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages171-190
Number of pages20
DOIs
StatePublished - Jan 1 2019

Publication series

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

Fingerprint

DNA Probes
Drosophila melanogaster
Fluorescence In Situ Hybridization
Drosophila
Fishes
Technology
Messenger RNA
Transcription Factors
Signal-To-Noise Ratio
Cardiac Myocytes
Nucleic Acids
Animal Models
DNA

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Blice-Baum, A. C., Vogler, G., Viswanathan, M. C., Trinh, B., Limpitikul, W. B., & Cammarato, A. R. (2019). Quantifying tissue-specific overexpression of FOXO in Drosophila via mRNA fluorescence in situ hybridization using branched DNA probe technology. In Methods in Molecular Biology (pp. 171-190). (Methods in Molecular Biology; Vol. 1890). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8900-3_15

Quantifying tissue-specific overexpression of FOXO in Drosophila via mRNA fluorescence in situ hybridization using branched DNA probe technology. / Blice-Baum, Anna C.; Vogler, Georg; Viswanathan, Meera C.; Trinh, Bosco; Limpitikul, Worawan B.; Cammarato, Anthony Ross.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 171-190 (Methods in Molecular Biology; Vol. 1890).

Research output: Chapter in Book/Report/Conference proceedingChapter

Blice-Baum, AC, Vogler, G, Viswanathan, MC, Trinh, B, Limpitikul, WB & Cammarato, AR 2019, Quantifying tissue-specific overexpression of FOXO in Drosophila via mRNA fluorescence in situ hybridization using branched DNA probe technology. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1890, Humana Press Inc., pp. 171-190. https://doi.org/10.1007/978-1-4939-8900-3_15
Blice-Baum AC, Vogler G, Viswanathan MC, Trinh B, Limpitikul WB, Cammarato AR. Quantifying tissue-specific overexpression of FOXO in Drosophila via mRNA fluorescence in situ hybridization using branched DNA probe technology. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 171-190. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8900-3_15
Blice-Baum, Anna C. ; Vogler, Georg ; Viswanathan, Meera C. ; Trinh, Bosco ; Limpitikul, Worawan B. ; Cammarato, Anthony Ross. / Quantifying tissue-specific overexpression of FOXO in Drosophila via mRNA fluorescence in situ hybridization using branched DNA probe technology. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 171-190 (Methods in Molecular Biology).
@inbook{0078f376b3f949ecb7c80d02989a8db8,
title = "Quantifying tissue-specific overexpression of FOXO in Drosophila via mRNA fluorescence in situ hybridization using branched DNA probe technology",
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.",
keywords = "bDNA, Branched DNA, Dorsal vessel, Drosophila melanogaster, FISH, Fluorescence in situ hybridization, Heart tube, RNAscope, ViewRNA",
author = "Blice-Baum, {Anna C.} and Georg Vogler and Viswanathan, {Meera C.} and Bosco Trinh and Limpitikul, {Worawan B.} and Cammarato, {Anthony Ross}",
year = "2019",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-8900-3_15",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "171--190",
booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Quantifying tissue-specific overexpression of FOXO in Drosophila via mRNA fluorescence in situ hybridization using branched DNA probe technology

AU - Blice-Baum, Anna C.

AU - Vogler, Georg

AU - Viswanathan, Meera C.

AU - Trinh, Bosco

AU - Limpitikul, Worawan B.

AU - Cammarato, Anthony Ross

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

KW - bDNA

KW - Branched DNA

KW - Dorsal vessel

KW - Drosophila melanogaster

KW - FISH

KW - Fluorescence in situ hybridization

KW - Heart tube

KW - RNAscope

KW - ViewRNA

UR - http://www.scopus.com/inward/record.url?scp=85056403280&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85056403280&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-8900-3_15

DO - 10.1007/978-1-4939-8900-3_15

M3 - Chapter

T3 - Methods in Molecular Biology

SP - 171

EP - 190

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -