Cytogenetic analysis of chromosomes provides the foundation for the clinical diagnosis of genetic disorders caused by chromosomal abnormalities. Initially, chromosome banding techniques such as G-banding were used as diagnostic tools. Banding techniques, however, lack the specificity and resolution needed for the diagnosis of a wide variety of chromosomal abnormalities.
Molecular cytogenetics (fluorescent in situ hybridization or FISH) combines classical cytogenetics with molecular techniques, which significantly increases the resolution and specificity of cytogenetic analysis. FISH with chromosome-specific probes reveal hybridization signals, even on inadequately spread metaphase chromosomes and interphase nuclei. It is capable of detecting subtle chromosomal abnormalities that are not detected using conventional cytogenetic analysis. Additionally, FISH allows quantitative analysis of target sequences, thus providing an advanced tool for genetic research.
During FISH, a fluorescent-labeled nucleic acid probe with sequences complementary to the area of interest on a chromosome is hybridized to chromosome spreads or nuclei. Probes bound to the target sequences are visualized by fluorescence microscopy.
During multicolor FISH, the use of fluorescent dyes with different emission wavelengths allows for the use of multiple probes during a single hybridization to simultaneously target different chromosomes or different areas within a chromosome. FISH can be qualitative (to study the presence, absence, or position of a sequence) or quantitative (e.g., to study the length of a repeat sequence).
In studies requiring quantitative measurement of a target sequence such as telomere-length measurement, it is critical to have slide-to-slide and day-to-day reproducibility to enable data comparability across various hybridizations. It is also crucial to have uniform hybridization to minimize the signal variations within a slide.
Current methodologies often utilize various sealants around a cover slip to create a sealed chamber, and hybridizations are incubated in a conventional oven. These methods generate data with high day-to-day variability in fluorescence intensity and are susceptible to photo-bleaching of fluorescent dyes.
In this article, we describe a new method for performing quantitative telomere FISH using the Hybex® microarray incubation system from SciGene (www.scigene.com). The Hybex hybridization chambers have advanced sealing to prevent loss of humidity and photo-bleaching, resulting in a more uniform hybridization and brighter signal, which are critical for quantitative FISH that requires high sensitivity and reproducibility. We have applied this procedure for molecular epidemiological studies and obtained highly reproducible results.
Metaphase chromosome spreads were prepared following standard cytogenetic procedures. Lymphocyte cultures were established by inoculating 1 mL of fresh blood into 9 mL of blood culture medium (RPMI-1640 medium supplemented with 15% bovine serum and 1.5% phytohemagglutinin). The cultures were incubated at 37ºC for four days. To arrest the cells at metaphase, 0.2 µg/mL colcemid was added to the culture one hour before harvest. The cells were treated in hypotonic solution (0.06 M KCl) at RT for 25 minutes.
The chromosome preparation from cultured lymphocytes were dropped onto a clean glass slide, fixed in 3:1 methanol:acetic acid for one hour at room temperature, and then air-dried. Slides were then dehydrated in an ethanol series (2 minutes each in 70%, 80%, 90%, and 100% ethanol) and then air-dried for 15 minutes.