Karyotyping (G-band)
Many of the genetic alterations found in cancer cells are microscopically visible when chromosomes condense during mitosis, and can thus be registered as numerical or structural cytogenetic abnormalities. Cytogenetic analysis is a well-established source of diagnostic and prognostic information for hematological malignancies and sarcomas, and it is also an important aid in the characterization of other solid tumors, even if the requirement of fresh tissue for culturing and the low rate of tumor cell proliferation in vitro represent substantial challenges to the latter.
Method Overview
To analyze the chromosomal contents of a given sample, fresh tissue must be obtained. Cells are then grown in tissue-culture flasks until they reach an optimal density, afterwhich a cell-cycle inhibitor is added to stop dividing cells at a stage in which nuclear chromatin is condensed into chromosomes. Cells are then detached from the flask (harvested), treated with a hypotonic solution to burst the plasma membrane and obtain isolated nuclei, placed in a fixative solution for preservation, and then dropped onto glass slides (spreading). At this point chromosomes are transparent and display no visible patterns, so a banding treatment is required, followed by a staining step. The most commonly used banding technique is Giemsa banding (G-banding), in which the metaphase spreads are exposed to a trypsin solution followed by the addition of a DNA binding dye (Giemsa or Wrights solution) to get the final pattern of black and white bands. Banded chromosomes can then be analyzed under a microscope to produce the sample karyotype, which consists of a standardized description of the chromosomal content of that sample.
Our group has hands-on experience in karyotyping by G-banding, but is currently using this technique only in characterization of cell lines.
G-banding of solid tumors is, in our collaborative projects, performed at the Department of Genetics of the Portuguese Oncology Institute, headed by Prof. Manuel Teixeira.
Method Overview
To analyze the chromosomal contents of a given sample, fresh tissue must be obtained. Cells are then grown in tissue-culture flasks until they reach an optimal density, afterwhich a cell-cycle inhibitor is added to stop dividing cells at a stage in which nuclear chromatin is condensed into chromosomes. Cells are then detached from the flask (harvested), treated with a hypotonic solution to burst the plasma membrane and obtain isolated nuclei, placed in a fixative solution for preservation, and then dropped onto glass slides (spreading). At this point chromosomes are transparent and display no visible patterns, so a banding treatment is required, followed by a staining step. The most commonly used banding technique is Giemsa banding (G-banding), in which the metaphase spreads are exposed to a trypsin solution followed by the addition of a DNA binding dye (Giemsa or Wrights solution) to get the final pattern of black and white bands. Banded chromosomes can then be analyzed under a microscope to produce the sample karyotype, which consists of a standardized description of the chromosomal content of that sample.
Our group has hands-on experience in karyotyping by G-banding, but is currently using this technique only in characterization of cell lines.
G-banding of solid tumors is, in our collaborative projects, performed at the Department of Genetics of the Portuguese Oncology Institute, headed by Prof. Manuel Teixeira.