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

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 number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, 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 intra-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. 0 1992 Wiley-Liss, Inc.

Expression of topoisomerase II alpha and beta in an adenocarcinoma cell line carrying amplified topoisomerase II alpha and retinoic acid receptor alpha genes

“…In situ hybridisation (Pinkel et al, 1986, Nederlof et al, 1992 Metaphase spreads were fixed in 3: 1 methanol, acetic acid for 1 h and then treated with RNAse (1I00 tg ml-') for 1 h. Slides were then fixed in 1% paraformaldehyde in PBS and dehydrated. Chromosomes were denatured prior to hybridisation in 70% formamide/2 x SSC at 70°C for 2 min and dehydrated.…”

Quantification of fluorescence in situ hybridization signals by image cytometry

“…For this purpose, specific hard-and software were developed for the automatic analysis of the ISH images. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities (16), as well as of fluorescence ratios obtained with double-labeled probes (17).…”

Fluorescence ratio measurements of double‐labeled probes for multiple in situ hybridization by digital imaging microscopy

“…On the other hand, preferential hybridization of one of the two labels will not occur when the two haptens are on the same molecule. The observation that the internuclear variation of the individual colors was low in this experiment should not be emphasized, since earlier studies (19) show that between experiments the CVs may vary from 35% to as much as 75%.…”