Bicolor fluorescence in situ hybridization to intron and exon mRNA sequences

Raap, Anton K.; Rijke, Frans M. van de; Dirks, Roeland W.; Sol, C. J. A.; Boom, R.; Ploeg, M. van der

doi:10.1016/0014-4827(91)90439-2

Cited by 61 publications

(40 citation statements)

References 19 publications

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“…Finally, the in situ hybridization method presented in this study proved to be compatible with riphery has been found to be directed by microfilaments (Sundell and Singer, 1991). On basis of these observations it has been suggested that a relationship exists between the localization of the mRNA and the functional location of the corresponding protein, although there are indications that this is not a general rule (Morales et al, 1991;Raap et al, 1991). With the advent of non-radioactive in situ hybridization it proved possible to detect defined RNA in situ hybridization signals in the nucleus, most likely showing active transcription of the gene and possibly the transport of its mRNA to the cytoplasm Lawrence et al, 1989).…”

Section: Introductionmentioning

confidence: 82%

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

Dirks

Dorp²,

Minnen³

et al. 1992

J Histochem Cytochem.

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The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea m g d s was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28s rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable presermaon of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphol-

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Section: Introductionmentioning

confidence: 82%

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

Dirks

Dorp²,

Minnen³

et al. 1992

J Histochem Cytochem.

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“…The pre-embedding EM ISH procedure was performed on polystyrene 6-well culture plates. The hybridization conditions were based on the LM ISH protocol previously described for cultured cells (40,41). A hybridization mixture, consisting of 60% deionized formamide, 2 x SSC (0.3 M NaCI.…”

Section: Methodsmentioning

confidence: 99%

Saponin pre-treatment in pre-embedding electron microscopic in situ hybridization for detection of specific RNA sequences in cultured cells: a methodological study.

Macville

Wiesmeijer²,

Dirks³

et al. 1995

J Histochem Cytochem.

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We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in situ hybridization (ISH). The specimens were monitored by reflection-contrast microscopy (RCM) before processing for EM. A good balance between preservation of ultrastructure and intensity of hybridization signals was obtained by using mild aldehyde furation followed by saponin permeabilization. Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (E) mRNA in rat 9G cells, and 28s rRNA in both cell lines. The hybrids were detected immunocytochemically by the peroxidaseldiaminobenzidhe (DAB) method or by ultra-small gold with silver enhancement. Comparison of these methods favored the peroxi-

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“…Not only a number of nuclear antigens, but also RNA transcripts have been found to accumulate in the nucleus (Lawrence et al 1989;Raap et al 1991). Following visualization by in situ hybridization, these RNA concentrations can be seen as punctate spots or track-like objects.…”

Section: T Cremer Is At the Institut Fur Humangenetik Und Anthropolomentioning

confidence: 99%

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

et al. 1993

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The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescence in situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocallaser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional networklike compartment, termed the interchromosome domain (ICO) compartment, in wh ich transcription and splicing of mRNA occurs.

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Bicolor fluorescence in situ hybridization to intron and exon mRNA sequences

Cited by 61 publications

References 19 publications

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

Saponin pre-treatment in pre-embedding electron microscopic in situ hybridization for detection of specific RNA sequences in cultured cells: a methodological study.

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

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