Question

Explain why fixed tissues can be used to perform fluorescence in situ hybridisation but not karyotyping. Compare what is being detected in each method and how samples are prepared.

Answer 1

Fluorescence in situ hybridization is a molecular technique used to detect chromosomal abnormalities and translocations by using a fluorescent probes that binds to their complementary nucleic acid sequence. It involves fixation and permeabilization of fixed cells/tissues followed by incubation with the fluorescent oligonucleotide probe that binds to its specific target (DNA/RNA) inside the cell. The cell or tissue sample can then be visualized under a fluorescent microsope to determine the expression or localization of specific DNA/ RNA targets.

Whereas karyotyping can only be performed on actively dividing cells that are arrested at metaphase stage by treatment with colchicine followed by spreading of these metaphase chromosomes on a glass slide. These chromosomes are the stained with giemsa stain and observed under a microscope to ascertain their banding pattern, relative size and numbers.

Since karyotyping requires induction of cells to divide and grow, hence it requires non fixed living tissue/cells. On the other hand FISH is used to detect specific RNA targets and can be done of formalin fixed tissues.