Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Raphael Pinaud, Claudio Mello, Tarciso A. Velho, Ryan D. Wynne, Liisa A. Tremere

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.

Original languageEnglish (US)
Pages (from-to)1370-1379
Number of pages10
JournalNature Protocols
Volume3
Issue number8
DOIs
StatePublished - 2008

Fingerprint

Fluorescence In Situ Hybridization
Brain
Haptens
Fluorescence
Tissue
Messenger RNA
Digoxigenin
Fluorophores
Biotin
Labeling
Peroxidase
Songbirds
Neurons
Sensory Receptor Cells
Genes
Population

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization. / Pinaud, Raphael; Mello, Claudio; Velho, Tarciso A.; Wynne, Ryan D.; Tremere, Liisa A.

In: Nature Protocols, Vol. 3, No. 8, 2008, p. 1370-1379.

Research output: Contribution to journalArticle

Pinaud, Raphael ; Mello, Claudio ; Velho, Tarciso A. ; Wynne, Ryan D. ; Tremere, Liisa A. / Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization. In: Nature Protocols. 2008 ; Vol. 3, No. 8. pp. 1370-1379.
@article{430071a9c8b54c38a3950e6316b1041f,
title = "Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization",
abstract = "Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.",
author = "Raphael Pinaud and Claudio Mello and Velho, {Tarciso A.} and Wynne, {Ryan D.} and Tremere, {Liisa A.}",
year = "2008",
doi = "10.1038/nprot.2008.115",
language = "English (US)",
volume = "3",
pages = "1370--1379",
journal = "Nature Protocols",
issn = "1754-2189",
publisher = "Nature Publishing Group",
number = "8",

}

TY - JOUR

T1 - Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

AU - Pinaud, Raphael

AU - Mello, Claudio

AU - Velho, Tarciso A.

AU - Wynne, Ryan D.

AU - Tremere, Liisa A.

PY - 2008

Y1 - 2008

N2 - Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.

AB - Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.

UR - http://www.scopus.com/inward/record.url?scp=50349095769&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=50349095769&partnerID=8YFLogxK

U2 - 10.1038/nprot.2008.115

DO - 10.1038/nprot.2008.115

M3 - Article

C2 - 18714305

AN - SCOPUS:50349095769

VL - 3

SP - 1370

EP - 1379

JO - Nature Protocols

JF - Nature Protocols

SN - 1754-2189

IS - 8

ER -