Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals

Authors

  • P. M. Nederlof,

    1. Sylvius Laboratory, Department of Cytochemistry and Cytometry, University of Leiden, 2233 Al Leiden, The Netherlands
    Current affiliation:
    1. Department of Laboratory Medicine, Division of Molecular Cytometry, MCB 230, University of California, San Francisco, San Francisco, CA 94143—0808
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  • S. van der Flier,

    1. Sylvius Laboratory, Department of Cytochemistry and Cytometry, University of Leiden, 2233 Al Leiden, The Netherlands
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  • A. K. Raap,

    Corresponding author
    1. Sylvius Laboratory, Department of Cytochemistry and Cytometry, University of Leiden, 2233 Al Leiden, The Netherlands
    • Sylvius Laboratory, Department of Cytochemistry and Cytometry, Universty of Leiden, Wassenaarseweg 72 2333 Al Leiden, The Netherlands
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  • H. J. Tanke

    1. Sylvius Laboratory, Department of Cytochemistry and Cytometry, University of Leiden, 2233 Al Leiden, The Netherlands
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  • This study was sponsored in part by the Netherlands Organization for Scientific Research (NWO) grant 534—060, and Pioneer Subsidie NWO grant 90—129.90.

Abstract

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy munber target sequences as a model system. These chromosome-specific probes were labeled with biotin, digogigenin, or fluorescein

Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH

The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation

The infra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified. Furthermore, an internal standard to correct for differences in hybridization efficiency among cells, based on a second hybridization, was introduced

It is concluded that development of an accurate quantitative ISH procedure requires further reduction of the internuclear variation. Results presented indicate that improvements in the preparation of the target material and in the hybridization reaction are especially needed to fulfill this requirement© 1992 Wiley-Liss, Inc.

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