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Volume 53 (3): 401-408, 2005
Copyright ©The Histochemical Society, Inc.
An Approach for Quantitative Assessment of Fluorescence In Situ Hybridization (FISH) Signals for Applied Human Molecular Cytogenetics
National Center of Mental Health, Russian Academy of Medical Sciences, Moscow, Russia (IYI,IVS,VVM,YBY), and Institute of Pediatrics and Children's Surgery, Russian Ministry of Health, Moscow, Russia (SGV)
Correspondence to: Y.B. Yurov, National Center of Mental Health, Russian Academy of Medical Sciences, Zagorodnoe sh.2, 119152, Moscow, Russia. E-mail: y_yurov{at}yahoo.com; i_yurov{at}mail.ru
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A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies. (J Histochem Cytochem 53:401–408, 2005)
Key Words: quantitative FISH • differentiation of FISH signals • aneuploidy scoring • low-level chromosomal • mosaicism • chromosome heteromorphism
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Introduction |
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FLUORESCENCE IN SITU HYBRIDIZATION (FISH) is considered to be the most commonly used molecular cytogenetic technique, having a wide variety of applications. Although it has been enhanced and modified since the early 1980s, there are a number of unsolved problems with the use of FISH for different diagnostic as well as research purposes. One of these is the interpretation of FISH results during chromosome identification using interphase FISH. In the study of aneuploidy by FISH in preimplantation and prenatal or postnatal diagnosis, the technique of quantitative signal differentiation appears to be required. This is of significant importance in FISH studies of low-level fetal and placental mosaicism and single blastomers or polar bodies because FISH artifacts can significantly reduce the resolution of the results. Over-position of interphase chromosome, somatic pairing of homolog chromosomes in nuclei, different replication patterns of chromosomes in dividing cells, or simply absence of signals of one or both homologous chromosomes due to low efficiency of hybridization are the most common reasons for the misinterpretation of FISH results. One possible solution to this problem is the development and application of methods for the quantitative analysis and differentiation of individual FISH signals from FISH artifacts.
Originally, quantitative analysis of fluorescent images was carried out for the improvement of routine cytogenetic tests (Pinkel et al. 1986
). Subsequently, quantitative studies of FISH images were introduced to investigations of tissue-level gene expression, transcriptional activation, coexpression, and nuclear structure function. Despite increased interest in the development of the approaches to FISH signal quantitative assessment, there is as yet no standardized or commonly used protocol. Briefly, the main difficulties of such studies are the lack of stable reproducibility, the irregularity of the signal, and background autofluorescence (reviewed by Levsky and Singer 2003). Therefore, development of adequate protocols for quantitative analysis of fluorescence images, especially for applied FISH studies, is an actual problem.
Here we present a relatively simple and rapid approach for the quantitative assessment of FISH signals based on the digital capturing of microscopic images and the intensity measuring of hybridization signals by Scion Image software originally developed for analyzing electrophoresis gels. We have carried out the tests for this technique studying aneuploidy and the low-level chromosomal mosaicism involving different human chromosomes in interphase nuclei of different tissues (chorionic villi, fetal skin, placenta, and neuronal cells of the adult brain). We have also investigated the efficiency of the approach in the differentiation of homologous chromosome heteromorphism by the quantitative analysis of alphoid DNA variation of chromosomes 13, 21, and X. In spite of many technical and theoretical limitations in the quantification of FISH signals, we were able to demonstrate the high reproducibility of the measurements after application of Scion Image software. We have concluded that this approach could be useful as an additional tool to visual microscopic analysis and to assist in correct FISH signal interpretation.
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Materials and Methods |
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Peripheral blood lymphocytes were obtained from patients with and without chromosome abnormality previously detected by routine cytogenetic studies. Metaphase spreads and interphase nuclei were prepared from blood lymphocytes obtained from these patients according to a standard protocol previously described in detail (Yurov et al. 1996
). The chorionic villi samples were obtained from the material of spontaneous abortions (5–10 weeks of gestation). These tissues were processed for FISH as follows: chorionic villi samples were washed in physiological solution three times. To clean the specimens of the rest of maternal deciduas and blood, samples were washed several times in 70% ethanol. They were then rinsed for 30 sec with 60% acetic acid and placed in solution of 60% acetic acid for 15–20 min at room temperature and periodically mixed by inversion. Dispersed single-cell suspensions were fixed in a methanol-acetic acid (3:1) fixative mixture two times for 30 and 50 min. The cells were dropped onto wet slides and air dried at room temperature. Three slides with two drops of cell suspensions each were prepared for each sample.
Fetal skin, placenta, and samples of postmortem adult brain tissues were processed as described previously (Yurov et al. 2001a). Fifty milligrams of these samples were homogenized in 2 ml of phosphate-buffered saline (PBS), pH 7.3, with 0.1% Nonidet P-40, using a glass Teflon homogenizer. Samples were centrifuged at 1000 x g for 5 min followed by fixation of pellets in an ice-cold methanol-glacial acetic acid mixture (3:1), three times for 20 min. The resulting suspensions of nuclei were then dropped onto wet slides. The slides were allowed to dry overnight and were dehydrated by an ethanol series before use in FISH experiments. The quality of the slides with the fixed nuclei was estimated according to the absence of surrounding cytoplasm; another fixation step of cell suspensions was performed, if necessary.
Permission from the Ethics Committee of the National Center of Mental Health, Russian Academy of Medical Sciences was obtained. Written informed consent was obtained from the patients for whom molecular cytogenetic studies were carried out.
The set of DNA probes from the original collection developed at the Laboratory of Cytogenetics, National Center of Mental Health, Moscow, Russia, and including chromosome enumeration DNA probes specific to chromosomes 1, 7, 8, 9, 13, and 21; 14 and 22; 15, 16, 18, X, and Y was used (Yurov et al. 1996,2002; Soloviev et al. 1998). FISH studies were performed as described in detail previously (Soloviev et al. 1994,1995; Yurov et al. 1996). For dual- and three-color hybridization, DNA probes were mixed in equal proportions (5 µl each probe at a concentration of 5 ng/µl for each probe). Labeled DNA probes were combined in the following order: (a) chromosome Y–specific probe (labeled by Cy3), chromosome X–specific probe (labeled by fluorescein-FluorX), chromosome 1–specific probe (labeled by biotin or AMCA); (b) chromosome 9–specific probe (labeled by biotin) and chromosomes 13/21–specific probe (labeled by Cy3); (c) chromosome 16–specific probe (labeled by biotin) and chromosomes 14/22–specific probe (labeled by Cy3); and (d) chromosome 15–specific probe (labeled by biotin) and chromosome 18–specific probe (labeled by Cy3). Hybridization was usually performed at 42C overnight, although clear hybridization signals were seen after 30–60 min of hybridization. The slides were washed in 50% formamide, 2 x SSC at 42–45C, three times for 2 min and rinsed in 0.1–2 x SSC for 5 min. Detection of biotin-labeled probes was performed as previously described (Pinkel et al. 1986) by the use of a layer of fluorescein-avidin (Sigma; Moscow, Russia). Slides were mounted in antifade solution [0.2% p-phenylenediamine (Sigma) in 80% glycerol, 20 mM TRIS-HCl, pH 8.0], and 200 ng/ml DAPI (4',6-diamidino-2-phenylindole-2HCl).
For epifluorescence microscopy, a Leitz Orthoplan microscope (Leica Mikroskopie und Systeme; Wetzlar, Germany) equipped with a 100 W lamp was used with the following filter sets (Leica Mikroskopie und Systeme): A (No. 513,596) for DAPI fluorescence; I3 (No. 513,719) or GR (No. 513,821) for fluorescein isothiocyanate signals; GR (No. 513,821) for both fluorescein and cyanine signals; N2 (No. 513,609) for cyanine signals. All images were observed with the Plan-Neofluotar x40/1.30 or x63/1.30–0.60 oil immersion lenses.
The relative intensity of FISH signals was obtained by digital capturing of microscopic image by the monochrome CCD camera (Cohu 4910 series; Cohu Inc., San Diego, CA), LG-3 grayscale scientific PCI frame grabber (Scion Corporation; National Institutes of Health, Frederick, MD), and subsequent measuring of the intensity of hybridization signals by Scion Image Beta 4.0.2 (Scion Corporation) acquired from www.scioncorp.com (accessed 12/07/2001). The quantification of FISH signals from each digital image was processed by the macros supplied by the manufacturer. Numerical values of the signal relative intensity were compared with each other in the case of interphase FISH study. For the homologous chromosome differentiation, the ratio of the signal relative intensity from the digital image was obtained and compared with the value from another digital image. The reproducibility of the intensity measuring was assessed by several quantitative analyses (5–10 times) of the same interphase nucleus or metaphase spread. All interphase nuclei suspected to have one signal were subjected to quantitative assessment of FISH signals. Ten to 20 metaphase spreads were analyzed for each sample.
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Results |
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Interphase FISH: Studies of Low-level Mosaicism and Somatic Chromosome Pairing
To test the efficiency of the proposed approach, we studied chromosome complement in interphase nuclei of chorionic villi, fetal skin, placenta, and neurons of the adult brain. Examples of quantification of FISH signals using Scion Image software are depicted in Figure 1. Examples of quantitative assessment of FISH signals in metaphase spreads and interphase nuclei are shown in Figure 2. The data demonstrate the high reproducibility of the measurement results, allowing the possibility of the differentiation of homologous chromosomes X, 21, and 16 by means of the study of alphoid or classical satellite DNA size heteromorphism in metaphase spreads and interphase nuclei.
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Interphase FISH analysis of 23 spontaneous abortion specimens (chorionic villi, fetal skin, and placenta) were performed using quantification of FISH signals. The results of the chromosome complement study using quantitative assessment of FISH signals in the samples with the mosaic form of monosomy are shown in Table 1. The technique applied has allowed us to confirm the mosaic form of monosomy of chromosomes 14/22, 15, 16, 18, X, and Y in 12 cases. In these studies, a confidence interval (threshold of percentage) of 95% for abnormal chromosome complement nuclei with scoring no less than 500 interphase nuclei was chosen. This means that no fewer than 25 nuclei (5%) from 500 analyzed nuclei should have one FISH signal indicating the presence of monosomy (mosaic case). In the present study of 12 spontaneous abortion specimens, an abnormal cell population varied from 6% to 93% (Table 1). The cells with the signal over-position in these specimens were present as well in low percentages (1–5%, mean value 3.2%). The approach has been successfully applied for discrimination of single signals with increased intensity ("pseudo-monosomy") due to over-position of heterochromatic regions (Figures 3A and 3B). Control samples (samples considered as normal, without regular cell population with abnormal chromosome complement) should have less than 5% of aneuploid cells involving target chromosomes in aberrant sample. In this study, monosomy was not confirmed for 11 control samples in fetal skin (seven specimens) and placenta (four specimens). In these samples, the average percentage of the FISH signal over-position ranged from 1.4% to 4.2%, with a mean value of 3.1%. The frequency of probable monosomic nuclei with the "normal" intensity of signals was less than 1%.
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We analyzed the chromosome complement in interphase nuclei of neurons in seven postmortem adult brain samples. DNA probes for chromosomes 1, 9, 13, 16, 18, 21, 22, X, and Y were applied. The regular forms of aneuploidy (including true monosomy) were not observed in any of the samples studied. However, a significant fraction of neuronal cell interphase nuclei had one signal per nucleus after hybridization with classical satellite DNA probes for heterochromatic regions of chromosomes 1, 9, and 16. The frequent appearance of one signal per nucleus in the brain tissues could be explained by the phenomenon of somatic pairing of homologous chromosomes shown previously for chromosomes 1 and 17 (Arnoldus et al. 1989
,1991). Taking this phenomenon into account, we performed quantitative analysis of signal intensities in nuclei with one and two signals. An example of such analysis is shown in a case of autopsy brain sample after hybridization with a chromosome 16–specific DNA probe (Figures 3C and 3D). Nuclei with one FISH signal have an approximately twofold increase of intensity compared to individual signals in nuclei with two FISH signals. The data show the high reproducibility of the measurement results in a case of chromosome 16–specific DNA probe, as well as for chromosomes 1– and 9–specific DNA probes (data not shown). Therefore, we can conclude that somatic pairing of heterochromatic regions of homologous chromosomes 1, 9, and 16 could occur in brain cells with increased frequencies. The phenomenon of somatic pairing of chromosomes 1, 9, and 16 was present in all of the brain specimens in 5% to 40% of nuclei (data not shown). The quantitative assessment of FISH signals in these samples was applied for identification of single signals with increased intensity due to somatic pairing of the homologous chromosomes; therefore, the lack of monosomy for the chromosomes mentioned above was confirmed.
Differentiation of Homologous Chromosome by FISH with Chromosome-specific DNA Probes
The differentiation of homologous chromosomes was performed in a metaphase spread of peripheral blood lymphocytes of Down syndrome family members using an alphoid DNA probe for chromosomes 13 and 21. The technique was also applied for differentiation of homologous chromosome X in females without chromosome abnormalities being an initial stage of the X-chromosome inactivation patterns studied by FISH (described previously in detail in Yurov et al. 2001b) (Figures 1C and 1D and Figures 2A and 2B). The stable reproducibility of the FISH signal quantitative assessment was difficult to achieve because of the irregularity of the signal and background autofluorescence, particularly when comparing different slides. To solve this problem, we applied the comparison of the ratio of the relative intensity of the signals from different digital images. We tested the approach in the studies of chromosome-21 nondisjunction in 15 families with Down syndrome offspring (affected child, mother, and father). The technique allowed us to determine the paternal origin of the additional chromosome 21 in 12 families (80%)—two paternal and 10 maternal; and in seven families (46.7%), it has allowed the determination of the meiosis stage of chromosome-21 nondisjunction (data not shown).
The differentiation of homologous chromosome X in females by FISH was found to be applicable to the molecular cytogenetic approach to the study of X-chromosome inactivation patterns. We studied 40 families (propositus and her mothers) with offspring affected by mental retardation not related to chromosome abnormality. An alphoid DNA probe specific for chromosome X was used. The example of quantitative assessment of variable chromosome X FISH signals used for differentiation of homologous chromosomes X is shown in Figure 1D and Figure 2A. The data show the high reproducibility of the measurement results, allowing the possibility of the differentiation of homologous chromosomes X by alphoid DNA heteromorphism. In 19 cases (47.5%), the approach of quantitative FISH signal assessment was found to be sufficiently efficient for the differentiation of homologous chromosomes X (data not shown).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Although there have been an increasing number of attempts at quantitative analysis of FISH signals, and an increased availability of a number of software programs for the registration, processing, and quantification FISH results, to date, there are no standardized or commonly recommended protocols for the quantitative analysis of FISH signals. As mentioned previously, in some routine diagnostic as well as research molecular cytogenetic studies, there is a special need for accurate FISH signal interpretation. The determination of FISH signal relative intensity or the relative intensity ratio could significantly improve the accuracy of data obtained. Many commercially available programs for FISH studies include capabilities for scoring or quantification of FISH signals. Details concerning protocols and efficiency of these FISH programs can be found in the descriptions of the software. We selected the software for image processing developed by the Scion Corporation for a number of reasons. First, this software is freely available and therefore can be used by anyone in FISH studies for non-commercial purposes. This means that results obtained can easily be reproduced by subsequent independent studies. In addition, Scion Image has been successfully applied for the quantification of electrophoresis gels in many laboratories (including our experience); therefore, positive results with this software application in FISH studies can be expected. Despite the fact that Scion Image application for FISH analysis has not been previously published, we decided to present our original results in this field. Our experience indicates that the quantification of FISH signals by Scion Image is a relatively simple and rapid approach for the quantitative assessment of FISH signals. The comparative analyses of the FISH signal relative intensity value in interphase nuclei of different types of tissues followed by discrimination of the over-posed (artifact) signals have been found to improve significantly the molecular cytogenetic scoring of chromosomal abnormalities. The reproducibility of the approach was assessed by five to ten analyses of the same interphase nucleus or metaphase spread. The variation of the signal relative intensity has been in the range of 20%, and is therefore adequate for quantitative comparison of FISH signals. The reproducibility of measurements between different cells of the same preparation is also high enough to be useful for the interpretation of signal scoring results.
It should be emphasized that in all the samples studied, over-position of signals was observed. As a result, the presence of a small but sufficient population of nuclei with one hybridization signal (instead of the two signals expected in a normal diploid cell) produces the problem of interpretation of the results. Additionally, the phenomenon of somatic pairing of homologous chromosomes observed with a higher frequency in brain tissues causes a misinterpretation of the aneuploidy FISH scoring. Therefore, the lack of an approach for signal discrimination could lead to misdiagnosis in the cases of low-level mosaicism. In the present paper, we show that digital quantification (rarely used in FISH analysis of aneuploidy) aids in avoiding the scoring of "pseudo-monosomy," the result of over-position (or somatic chromosome pairing), leading to more accurate study results. The application of the approach has allowed us to exclude or confirm monosomy in the samples studied. Our results show that a confidence interval in interphase FISH studies of aneuploidy could probably be significantly less than 95%. Therefore, on the basis of the data we have obtained, we conclude that this approach is efficient enough for studies of low-level chromosomal mosaicism in different tissues.
An additional application of the proposed approach is the differentiation of homologous chromosome parental origin. Homologous chromosomes in normal chromosomal complement usually have no morphological differences (with the exception of acrocentric chromosomes with variable short arms or heteromorphism of chromosomes 1, 9, and 16 after C-banding). However, variations of alpha satellite DNA present in the centromeric regions of all human chromosomes can be visualized by in situ hybridization with chromosome-specific alphoid DNA probes (Yurov et al. 1987,2001b). In the present study, we analyzed the efficiency of the quantitative assessment of FISH signals for determining the parental origin of the additional chromosome 21 as well as differentiation of homologous chromosome X. The main difficulty of this study was that we had to compare the intensity of signals from the different slides. For more adequate comparative analysis, we proposed to use the ratio of relative intensity of FISH signals for chromosome 13 and 21 in the cases of chromosome 21 nondisjunction assays and chromosome-X signals in the case of X-chromosome inactivation study. We hypothesized that despite the variation of signal intensity from different slides, the ratio would not vary significantly, inasmuch as the ratio of the heterochromatin block size in homologous chromosomes is the same. The data obtained indicate that quantitative assessment of FISH signals is efficient up to 80% in the study of chromosome-21 nondisjunction. It should be emphasized that molecular cytogenetic studies are strongly recommended for more accurate cytogenetic diagnosis of trisomy 21, and therefore it would be quite helpful to determine the parental origin of the additional chromosome 21 and the meiosis stage of nondisjunction during a routine FISH analysis of a family with offspring affected by Down syndrome.
Initially, the molecular cytogenetic technique based on the identification of heteromorphism of homologous X chromosomes was found to sufficient for the study of X-chromosome inactivation patterns in Rett syndrome (Yurov et al. 2001b). However, the frequency of chromosome X with clearly visible heteromorphism after FISH with an alphoid DNA probe was found to be rather uncommon (in 8/33 individuals analyzed), probably due to the difficulty of comparative visual analysis of FISH signals. We propose the differentiation of active and inactive chromosome X with the quantitative assessment of FISH signals after application of a chromosome X–specific alphoid DNA probe for improving significantly the efficiency of the molecular cytogenetic technique used in the study of X-chromosome inactivation. This assay is useful in X-chromosome inactivation studies in females with mosaic forms of the chromosome-X aneuploidy, because molecular genetic techniques are unable to precisely determine X-chromosome inactivation patterns in these cases. We have shown the application of the quantitative assessment of FISH signals for chromosome X to increase the efficiency of this technique up to 47.5%, based on the registration of chromosomal heteromorphism with low differences in alphoid DNA content between homologous chromosomes. Moreover, the results of initial molecular cytogenetic assays for X inactivation were in full agreement with the results of subsequent analysis of the same cases by means of a restriction/quantitative PCR–based assay (AR-assay), a commonly used technique for X-chromosome inactivation studies (Iourov et al. 2003). We believe that this approach for the rapid quantitative assessment of FISH signals using Scion Image software will help in molecular cytogenetic studies of human chromosomes in different fields of research, including preimplantation, and pre- and postnatal diagnosis of aneuploidies by interphase FISH. This approach can be considered a highly efficient additional tool for use in studies of chromosome number variations in interphase nuclei, with a unique possibility for identification of aneuploidy with low-level chromosomal mosaicism.
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Acknowledgments |
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Supported by Copernicus 2 grant no. ICA2-CT-2000-10012 and INTAS grant no. 03-55-4060.
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Footnotes |
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Presented in part at the 14th Workshop on Fetal Cells and Fetal DNA: Recent Progress in Molecular Genetic and Cytogenetic Investigations for Early Prenatal and Postnatal Diagnosis, Friedrich Schiller University, Jena, Germany, April 17–18, 2004.
Received for publication May 29, 2004; accepted September 23, 2004
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