Q-FISH

Quantitative Fluorescent in situ hybridization (Q-FISH) is a cytogenetic technique based on the traditional FISH methodology.

Q-FISH is most commonly used to study telomere length, which in vertebrates are repetitive hexameric sequences (TTAGGG) located at the distal end of chromosomes.

This medium generally contains DAPI (a DNA counterstain) and an antifade solution to preserve the PNA fluorescence and reduce photobleaching.

In addition to the fluorescent reference beads, signal strength from sister chromatids should be equal and therefore can be used as another control to gauge the precision of the data.

Conversely, unlike Q-FISH, flow-FISH is unable to determine telomere length in a particular chromosome within an individual cell.

Multiplex-FISH uses a variety of probes to visualize the 24 chromosomes in different colours and identify intra- or inter-chromosomal rearrangements.

The relation between centromere abnormalities or chromosomal rearrangements and telomere length may have high clinical impact, since all appear important in pre- or post-natal diagnostics and tumor developments.

Observing the regulation of telomere length in different species also reveals important information about karyotype evolution and relevance to human illnesses.

[2] In another example, the non-homologous end joining (NHEJ) protein repairs double-stranded DNA breaks and relies on the Ku70/Ku80 heterodimer to function.

For example, in mice lacking the Ku 80 gene, the telomere lengths are measured by qFISH and are observed to be significantly shorter.

Telomere shortening causes genomic instability and occurs naturally with advanced age, both factors that correlate with possible causes of cancer.

In addition, the method provides greater resolution, allowing the user to examine the telomere length of each individual chromosome (p or q arm) in a particular cell.

The technique depends on well-prepared metaphase cells and it is vital that the equipment and samples are adjusted/normalized correctly in order for the quantification to be accurate.

Similarly, if one is using different cell types, many of the steps in Q-FISH (such as the length of colcemid treatment) will require optimization.

[1] A common problem in fluorescence microscopy is photobleaching, where the fluorophore loses its activity as a result of exposure to light.

[1] Prior to the development of Q-FISH and PNAs, the classical technique for measuring telomere length was the use of Southern blots.

Combined with traditional gram staining of positive blood cultures, PNAs can be used to target species-specific rRNA (ribosomal RNA) to identify different strains of bacteria or yeast.