Radio-Isotopic In Situ Hybridization on Tissue Sections: Practical Aspects and Quantification

Moorman, Antoon F.M.; Boer, P A de; Hagoort, Jaco; Franco, Diego; Lamers, W. H.

doi:10.1385/1-59259-066-7:97

Cited by 21 publications

(28 citation statements)

References 7 publications

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“…Adjacent series of 4 sections were stained by in situ hybridization with [35] S-labeled complementary RNA probes of glutamine synthetase (GS), 12 phosphoenolpyruvate carboxykinase (PEPCK), 13 ornithine aminotransferase (OAT), 14 or carbamoylphosphate synthetase (CPS), 15 exactly as described. 16 The sections were simultaneously processed under well-defined conditions to ensure that the optical density (OD) of the autoradiography signals linearly reflected the cellular messenger RNA levels (mRNA). 16,17 The maximum OD did not exceed 0.7.…”

Section: Methodsmentioning

confidence: 99%

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Stereological measurement of porto-central gradients in gene expression in mouse liver

et al. 2004

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The liver is thought to consist of lobules, numerous repeating, randomly oriented units. Within these lobules, genes are expressed in gradients along the porto-central axis, which spans the distance between portal and central veins. We have developed a robust stereological method to map all points in an image to their position on this porto-central axis. This approach is based on the distribution of well-characterized periportal and pericentral enzymes, which are visualized on sections preceding and following the section of interest. Because expression of the model genes phosphoenolpyruvate carboxykinase and ornithine aminotransferase declines gradually with increasing distance from the portal vein and central vein, respectively, these genes can be used to prepare images with topographical information without any assumption about the shape of the hepatic unit, or about the direction or shape of the gradient to be determined. The "relative distance" image is a 2-dimensional image that accurately maps the relative position of hepatocytes on the porto-central axis in 3-dimensional space. It is superimposed on the serial section under investigation to relate local staining density to position on the porto-central axis and obtain the gene expression gradient. The method was used to determine the expression gradient of 2 periportal and 2 pericentral enzymes and their response to fasting. The "total distance" image was used to measure the length of the porto-central axis, which was approximately 210 m in mice and found to decrease 13% after 1 day of starvation. The method can be applied to any tissue component that can be stained quantitatively. (HEPATOLOGY 2004;39:343-352.)

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Section: Methodsmentioning

confidence: 99%

“…16 The sections were simultaneously processed under well-defined conditions to ensure that the optical density (OD) of the autoradiography signals linearly reflected the cellular messenger RNA levels (mRNA). 16,17 The maximum OD did not exceed 0.7.…”

Section: Methodsmentioning

confidence: 99%

Stereological measurement of porto-central gradients in gene expression in mouse liver

et al. 2004

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“…In situ hybridization was carried out exactly as described (Moorman et al 2000). In situ hybridization carried out according to this protocol produces semiquantitative data, that is, differences in absorbance between different tissues or cells within one section reflect differences in mRNA concentration between these tissues or cells (Jonker et al 1997).…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Expression of thyroid hormone receptors A and B in developing rat tissues; evidence for extensive posttranscriptional regulation

Keijzer

Blommaart²,

Labruyère³

et al. 2007

Journal of Molecular Endocrinology

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The perinatal changes in the pattern of expression of the thyroid hormone receptor (TR) isoforms TRa 1 TRa 2 , TRb 1 , and TRb 2 were investigated using in situ hybridization and immunohistochemistry, and RT-PCR and western blotting as visualization and quantification techniques respectively. In liver, lung, and kidney, TRa mRNA was expressed in the stromal and TRb mRNA in the parenchymal component of the tissues. When compared with liver, TRa mRNA concentrations were tenfold higher in lung, kidney, and intestine, and 100-fold higher in brain, with TRa 2 mRNA concentrations exceeding those of TRa 1 5-to 10-fold. Tissue TRb 1 mRNA concentrations were similar in liver, lung, and brain, and 3-to 5-fold higher in kidney and intestine. None of the TRb 2 mRNA could be detected outside the pituitary. Tissue TRa 2 and TRb 1 protein levels reached adult levels at 5 days before birth, whereas TRa 1 protein peaked after birth. Because of the distinct time-course of thyroid hormonebinding receptors TRa 1 and TRb 1 , we speculate that an initiating, TRb 1 -mediated signaling from the parenchyma is followed by a TRa 1 -mediated response in the stroma. When compared with organs with a complementary parenchymal-stromal expression pattern, organs with extensive cellular co-expression of TRa and TRb (brain and intestinal epithelium) were characterized by a very low TRa protein: mRNA ratio, implying a low translational efficiency of TR mRNA or a high turnover of TR protein. The data indicate that the TR-dependent regulatory cascades are controlled differently in organs with a complementary tissue expression pattern and in those with cellular co-expression of the TRa and TRb genes.

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“…Gray-scale radioactive in situ hybridisation signals were converted into colour-scaled images to easily identify quantitative differences in gene expression within different myocardial compartments, using the methodology described by Moorman et al (2000).…”

Section: Image Analysismentioning

confidence: 99%

Species‐specific differences of myosin content in the developing cardiac chambers of fish, birds, and mammals

et al. 2002

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Key morphogenetic events during heart ontogenesis are similar in different vertebrate species. We report that in primitive vertebrates, i.e., cartilaginous fishes, both the embryonic and the adult heart show a segmental subdivision similar to that of the embryonic mammalian heart. Early morphogenetic events during cardiac development in the dogfish are long-lasting, providing a suitable model to study changes in pattern of gene expression during these stages. We performed a comparative study among dogfish, chicken, rat, and mouse to assess whether species-specific qualitative and/or quantitative differences in myosin heavy chain (MyHC) distribution arise during development, indicative of functional differences between species. MyHC RNA content was investigated by means of in situ hybridisation using an MyHC probe specific for a highly conserved domain, and MyHC protein content was assessed by immunohistochemistry. MyHC transcripts were found to be homogeneously distributed in the myocardium of the tubular and embryonic heart of dogfish and rodents. A difference between atrial and ventricular MyHC content (mRNA and protein) was observed in the adult stage. Interestingly, differences in the MyHC content were observed at the tubular heart stage in chicken. These differences in MyHC content illustrate the distinct developmental profiles of avian and mammalian species, which might be ascribed to distinct functional requirements of the myocardial segments during ontogenesis. The atrial myocardium showed the highest MyHC content in the adult heart of all species analysed (dogfish (S. canicula), mouse (M. musculus), rat (R. norvegicus), and chicken (G. gallus)). These observations indicate that in the adult heart of vertebrates the atrial myocardium contains more myosin than the ventricular myocardium. Anat Rec 268: 27-37, 2002.

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Radio-Isotopic In Situ Hybridization on Tissue Sections: Practical Aspects and Quantification

Cited by 21 publications

References 7 publications

Stereological measurement of porto-central gradients in gene expression in mouse liver

Stereological measurement of porto-central gradients in gene expression in mouse liver

Expression of thyroid hormone receptors A and B in developing rat tissues; evidence for extensive posttranscriptional regulation

Species‐specific differences of myosin content in the developing cardiac chambers of fish, birds, and mammals

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