Genes and Environment Laboratory, University of California, Berkeley
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Ji, Z., Zhang, L. Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay. J. Vis. Exp. (60), e3619, doi:10.3791/3619 (2012).
Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis.
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously.
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.
1. Sample slide preparation
2. Localization of metaphases using an automated system
4. Mounting and visualization of results
5. Representative Results
Figure 3A shows a normal metaphase cell in Square 2. (1)-(3): Chromosomes 8, 21 and 12 were painted red, green, and blue, and are visualized through a Texas Red, FITC, and DEAC filter, respectively; (4): Visualization through a DAPI/FITC/Texas Red triple filter. Generally, the chromosomes painted with blue are not clear through the triple filter and need to be viewed under the specific DEAC filter.
Figure 3B shows representative abnormal cells with leukemia-specific chromosomal translocation and aneuploidy. (1): t(8;21), a common chromosomal translocation in acute myeloid leukemia; (2): Trisomy 21, three copies of chromosome 21, a common aneuploidy in leukemia.
Figure 1. Arrangement of chromosome combinations on the OctoChrome device and expected chromosomal painting results.
Figure 2. Positioning of sample slide over the OctoChrome device.
Figure 3. Representative results obtained from OctoChrome FISH (Square 2).
Figure 3A. A normal cell with chromosomes 8, 12, 21 painted in Square 2.
Figure 3B. Representative abnormal cells with leukemia-specific chromosomal translocation and aneuploidy in Square 2.
This novel OctoChrome-FISH assay allows one to simultaneously examine all 24 human chromosomes in a single hybridization on one slide. The arrangement of chromosome combinations on the 8 squares has been designed to facilitate the identification of the non-random chromosome rearrangements in the most common leukemias and lymphomas. Thus, the assay can detect numerical (aneuploidy) as well as structural (translocations) chromosome alterations concurrently.
The most critical step in this assay is controlling the humidity during the overnight hybridization in the floating chamber in the water bath. The FISH probes and hybridization buffer easily dry out if the humidity is low, or become too dilute by absorbing too much moisture if the humidity is high. Either outcome could adversely impact the results. A possible modification to overcome this challenge is to seal the OctoChrome device and sample slide with tape and hybridize on a 37°C hotplate in the dark overnight.
Sample slides that have been aged too much may be unsuitable for use in this assay directly. While digesting such samples with pepsin will improve hybridization, storing prepared sample slides in a nitrogen atmosphere at -20°C is strongly recommended to minimize aging.
OctoChrome-FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma. It is also very useful for examining most specific chromosomal rearrangements related to human leukemia and lymphoma in populations exposed to potential leukemogens and lymphomagens and for studying the aneuploidy-inducing effects of chemicals in a chromosome-wide manner. Our previous CWAS study using this technique demonstrated that certain chromosomes may be more affected than others by exposure to benzene8,10, a primary industrial chemical and a ubiquitous environmental pollutant that causes human leukemia14. This phenomenon of “selective aneuploidy” could be explored in other chemical exposures using CWAS.
We have nothing to disclose.
The authors would like to thank Dr. Cliona M. McHale for critically reading and editing the manuscript. This work was funded by the National Institute of Environmental Health Sciences, National Institute of Health grant R01ES017452 to L Zhang.
|OctoChrome Kit||Cytocell Ltd.||PMP 803|
|Carnoy’s fixative||Methanol : glacial acetic acid = 3: 1|
|Fluorescence microscope||Equipped with filters to view DAPI, Texas Red, FITC, and Coumarin spectra individually and a DAPI/FITC/Texas Red triple filter to view different colors simultaneously|
|Metafer software||MetaSystems, Altlussheim, Germany||Facilitate to locate all metaphases and to re-evaluate the abnormalities|