CGH and Telomere Measurement
Quantification of DNA content in chromosomes.
Comparative genomic hybridization (CGH) is a technique for analyzing copy number variations relative to ploidy levels in the DNA of a test sample compared to a reference sample. This is achieved through the use of comparative fluorescence in situ hybridization. CGH is often used for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue. Chromosome-based CGH can be done easily with a dedicated CGH module for Isis.
Telomeres are regions of repetitive sequences at each end of a chromosome, protecting the end of the chromosome from deterioration or from fusion with neighboring chromosomes. The length of telomeres is subject to many studies, as it is believed to be related to ageing and cancer formation. Isis is capable of evaluating telomere signals based on their intensities.
The software for comparative genomic hybridization (CGH) analysis is available as an upgrade for Isis. CGH allows the detection of unbalanced copy number changes (gains and losses) of a given genomic DNA. The sample DNA and normal reference DNA are labelled with different fluorochromes. Both samples are mixed and co-hybridized onto normal metaphase chromosomes. Quantitative analysis of the color ratio along the axis of each chromosome yields the differences of the DNA copy number between sample and reference DNA. In Isis, metaphases can be easily karyotyped, and each karyotype can be immediately checked with control profiles. The analysis is done by averaging all chromosomes of a given class over all karyograms of the case, or even over different cases. Significance limits for the profiles can be set as fixed limits, as multiples of the standard deviation, or as probability limits. The simultaneous gain/loss display of different cases allows direct comparison of patients or preparations in order to detect common gains and losses.
Hybridization quality is always crucial for CGH. For reliable reproducibility of results, the CGH image quality is visualized in a ratio histogram and serves as a measure for the quality of your preparation. Chromosomes of the same class with a different condensation rate are straightened up and normalized to a uniform length, which enhances accuracy of the CGH results. High resolution sampling along the chromosome axes assures the detection of subtle chromosomal imbalances as small as 3 Mbp.
Cells showing artefacts can be excluded interactively. The individual profiles of each chromosome can be highlighted to identify outlier profiles. Once outlier profiles have been identified, the corresponding metaphase can be temporarily excluded from the analysis. The average profiles of tumor and reference DNA are visualized for each chromosome class including the number of assigned chromosomes and can be calculated across the same or different cases. Combined bars highlighting gains or losses can be activated and are shown next to the idiograms. This facilitates identifying gain/loss regions which the DNAs under examination have in common.
Telomeres of chromosomes labelled with a pan-telomeric FISH probe can be automatically analyzed with Isis. Quantifying the amount of telomeric DNA in cancer specimens is an area of growing interest in cytogenetics, since telomeres are essential for the stability and the replication of chromosomes. Telomeres are also thought to play an important role in senescence and apoptosis.
With Isis, telomere signals are automatically identified and analyzed for their intensity. The measurement regions can be dynamically set with the mouse. Reference signals such as centromere probes can be obtained either in the same color channel like the telomere signals, or using a different fluorochrome. For normalization of results, telomere analysis data are related to the reference signal intensity. Hence, variations in hybridization efficiency between individual metaphases can be compensated. Intensities for each pixel are corrected by the background intensity, and during the measurement the intensity is summarized for the telomere area.
Results from the telomere measurement are automatically stored in text files. They can be displayed on screen and are summarized for all analyzed karyograms. Data are displayed either as a table or as a graphic representation.