Chapter 2 Localization of mRNAs by in Situ Hybridization

doi:10.1016/S0091-679X(08)60568-3

Methods in Cell Biology

Volume 35, 1991, Pages 37-71

https://doi.org/10.1016/S0091-679X(08)60568-3 Get rights and content

Publisher Summary

This chapter discusses the localization of messenger RNAs (mRNAs) by in situ hybridization. In situ hybridization helps in distinguishing the cells in a complex tissue that express specific mRNAs and in making semiquantitative estimates of the relative concentration of these in different cell types. Other methods of analysis of RNA isolated from cells, tissues, or organisms—such as RNA blots or solution hybridization—average the content of a specific mRNA over all cell types in the sample. In situ hybridization offers several other potential advantages over solution or filter hybridization methods. The aim and many of the advantages of in situ hybridization are shared with the immunocytochemical detection of the encoded proteins. The basic difference is that they monitor gene expression at different levels. By measuring mRNA concentrations, a more accurate estimate of gene activity is provided because mRNA accumulates before the protein it encodes, and because relatively stable proteins may persist long after the gene is repressed and the mRNA has decayed. The technique described in the chapter employs the hybridization of radiolabeled riboprobes to sections of aldehyde-fixed and paraffin-embedded tissue.

References (52)

G.L. Ada et al.
Correlation of grain counts with radioactivity (¹²⁵1 and ³H) in autoradiography
Exp. Cell Res.
(1966)
L. Angerer et al.
Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo
Dev. Biol.
(1985)
L.M. Angerer et al.
Spatial patterns of metallothionein mRNA expression in the sea urchin embryo
Dev. Biol.
(1986)
L.M. Angerer et al.
Identification of tissue-specific gene expression by in situ hybridization
D.J. Brigati et al.
Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes
Virology
(1983)
R.J. Britten et al.
Analysis of repeating DNA sequences by reassociation
K.H. Cox et al.
Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes
Dev. Biol.
(1984)
K.H. Cox et al.
Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis
J. Mot. Biol.
(1986)
P.E. Hardin et al.
The Spec2 genes of Strongylocentrotus purpuratus: Structure and differential expression in embryonic aboral ectoderm cells
J. Mol. Biol.
(1988)
M.A. Harkey et al.
Coordinate accumulation of primary mesenchyme-specific transcripts during skeletogenesis in the sea urchin embryo
Dev. Biol.
(1988)

B.R. Hough-Evans et al.

Correct cell type-specific expression of a fusion gene injected into sea urchin embryos

Dev. Biol.

(1987)

J.B. Lawrence et al.

Intracellular localization of messenger RNA for cytoskeletal proteins

Cell (Cambridge, Mass.)

(1986)

M.C. Rykowski et al.

Precise determination of the molecular limits of polytene chromosome band regulatory sequences for the Notch gene are in the interband

Cell (Cambridge, Mass.)

(1988)

M.H. Stoler

In situ hybridization

K. Taneja et al.

Use of oligodeoxynucleotide probes for quantitative in situ hybridization to actin mRNA

Anal. Biochem.

(1987)

D.G. Wilkinson et al.

Expression of the proto-oncogene int-1 is restricted to specific neuronal cells in the developing mouse embryo

Cell (Cambridge, Mass.)

(1987)

Q. Yang et al.

Structure and tissue-specific developmental expression of a sea urchin arylsulfatase gene

Dev. Biol.

(1989)

M.E. Akam et al.

The distribution of Ultrabiothorax transcripts in Drosophila embryos

EMBO J.

(1985)

L.M. Angerer et al.

Detection of polyA RNA in sea urchin eggs and embryos by quantitative in situ hybridization

Nuclei Acids Res.

(1981)

L.M. Angerer et al.

In situ hybridization with ³⁵S-labeled RNA probes

Biotech. Update

(1989)

L.M. Angerer et al.

In situ hybridization with RNA probes—an annotated recipe

L.M. Angerer et al.

Progressively restricted expression of a homeobox gene within aboral ectoderm of developing sea urchin embryos

Genes Dev.

(1989)

C.E. Bandtlow et al.

Cellular localization of nerve growth factor synthesis by in situ hybridization

EMBO J.

(1987)

S.A. Berman et al.

Localization of an acetylcholine receptor intron to the nuclear membrane

Science

(1990)

M. Brahic et al.

Detection of viral sequences of low reiteration frequency by in situ hybridization

Proc. Natl. Acad. Sci. U.S.A.

(1978)

Cited by (88)

The recommended protocol for in situ hybridization
2020, In Situ Molecular Pathology and Co-expression Analyses
Up until this chapter, none of this book has been a “cookbook” of how to use in situ hybridization or immunohistochemistry. I developed the book this way because of my strong belief that the stronger foundation you have in the theory of the methodologies, the better you will be able to develop your own protocol and troubleshooting strategy. This chapter presents my cookbook recipe to do in situ hybridization. It can serve as a starting point for you to develop your own protocol. The protocol in this chapter stresses several key points. They include the importance of using several different concentrations of the probe and varying the pretreatment conditions. This strategy will increase the likelihood that you will find the optimal conditions for the detection of RNA or DNA for any given tissue. Another key point stressed in this chapter is the importance of monitoring the in situ reaction so as to maximize signal and reduce background.
The basics of in situ hybridization
2020, In Situ Molecular Pathology and Co-expression Analyses
The biochemical differences between tissues fixed with cross-linking fixatives (formalin, paraformaldehyde, glutaraldehyde) and those fixed in denaturing fixatives (Carnoy’s, acetone, alcohols, and acetic acid) or unfixed samples are profound. An understanding of this point allows you to better understand the marked differences in signal strength, subcellular localization, and coexpression capability when comparing these two broad categories of tissues. This chapter takes you on a journey through each step used during in situ hybridization and focuses on the biochemical explanations regarding why variables such as the type of probe, the type of fixative, and the wash are so important for optimal results. Further, this chapter introduces the phenomenon that different formalin-fixed, paraffin-embedded tissues, even if they share the same exact histologic diagnosis, may present very different three-dimensional biochemical profiles, which can have a strong impact on the optimal pretreatment condition for in situ hybridization.
Brahman genetics influence muscle fiber properties, protein degradation, and tenderness in an Angus-Brahman multibreed herd
2018, Meat Science
Citation Excerpt :
Immunohistochemistry was used to detect myosin heavy chain (MHC) types I, IIa and IIx, and determine fiber cross-sectional area (CSA). Cross sections (10 μm) of LL were cut on a cryostat (Microm HM 525, Tritech Inc., Edgewater, MD), placed on silanized slides (Angerer & Angerer, 1991), and stored at − 80 °C until subsequent analysis. Sections were blocked in 1 × PBS containing 5% (v/v) goat serum for 1 h at room temperature.
The objective of this study was to determine the influence of Brahman genetics on muscle contractile and metabolic phenotype and postmortem proteolysis. Cattle used in this study represent a continuous spectrum of Angus-Brahman genetic variation. Steers were harvested and Longissimus samples were collected at 1.5 h, 24 h, and 14 d postmortem. Proteolysis during the 14d aging period was evaluated, along with Warner-Bratzler shear force (WBSF) and trained sensory panel tenderness. Myosin heavy chain composition and enzymatic activity were used to evaluate fiber type characteristics. As Brahman influence increased, WBSF increased and sensory tenderness decreased. Calpain-1 autolysis decreased as Brahman percentage increased, and corresponded with reduced degradation of troponin-T, desmin, and titin. Increasing Brahman percentage was associated with greater citrate synthase activity and greater cross-sectional area of type IIx fibers. Brahman-influenced cattle produced tougher steaks and exhibited decreased protein degradation. Thus, Brahman genetics impacted not only the calpain-calpastatin system, but also muscle fiber size and metabolic properties.
Molecular biology techniques in musculoskeletal research
2006, Equine Surgery
Molecular Biology Techniques in Musculoskeletal Research
2005, Equine Surgery, Third Edition
Water-dispersible fluorescent nanospheres from poly(solketal acrylate)-block-poly(2-hydroxyethyl acrylate)
2004, Polymer
Reported in this paper is the preparation of fluorescent nanospheres from poly(solketal acrylate)-block-poly(2-hydroxyethyl acrylate) or PSA-PHEA. The strategy involved the chemical derivation of the PHEA block to graft the fluorophore fluorosceinamine (Fl) first. A selective solvent for PSA was then used to induce micelle formation from the diblock with PSA as the corona and the fluorescein-tagged PHEA block as the core. Nanospheres with fluorescent cores were obtained after PHEA core crosslinking with succinyl chloride. Water-dispersible nanospheres were prepared after removing the acetonide groups from the PSA block by hydrolysis to yield poly(glyceryl acrylate). Such nanospheres may find applications in fluorescent in situ hybridization assays.

View all citing articles on Scopus

View full text

Chapter 2 Localization of mRNAs by in Situ Hybridization

Publisher Summary

Exp. Cell Res.

Dev. Biol.

Dev. Biol.

Virology

Dev. Biol.

J. Mot. Biol.

J. Mol. Biol.

Dev. Biol.

Dev. Biol.

Cell (Cambridge, Mass.)

Cell (Cambridge, Mass.)

Anal. Biochem.

Cell (Cambridge, Mass.)

Dev. Biol.

The distribution of Ultrabiothorax transcripts in Drosophila embryos

EMBO J.

Detection of polyA RNA in sea urchin eggs and embryos by quantitative in situ hybridization

Nuclei Acids Res.

In situ hybridization with 35S-labeled RNA probes

Biotech. Update

In situ hybridization with RNA probes—an annotated recipe

Progressively restricted expression of a homeobox gene within aboral ectoderm of developing sea urchin embryos

Genes Dev.

Cellular localization of nerve growth factor synthesis by in situ hybridization

EMBO J.

Localization of an acetylcholine receptor intron to the nuclear membrane

Science

Detection of viral sequences of low reiteration frequency by in situ hybridization

Proc. Natl. Acad. Sci. U.S.A.

In situ hybridization with ³⁵S-labeled RNA probes