QFISH

qFISH, short for quantitative fluorescence in situ hybridization, is a cytogenetic technique that combines fluorescence in situ hybridization with quantitative image analysis to measure the amount of a specific DNA sequence in individual cells or chromosomes. The method uses fluorescent probes, often peptide nucleic acid (PNA) probes, that hybridize to repetitive sequences such as telomeres or to specific chromosomal loci. After hybridization, the fluorescence intensity of each signal is measured by microscopy, and signals are normalized against an internal reference signal or an external standard to convert intensity into a quantitative estimate of sequence length or copy number.

Telomere length measurement is a prominent application (often termed Telo-qFISH). In this context, telomere signals are compared to reference signals to obtain an estimate of telomere length in kilobases or relative units; measurements can be performed on metaphase chromosome spreads or on interphase nuclei.

qFISH allows analysis at single-cell and single-telomere resolution, enabling comparisons across cell types, disease states, or treatment conditions. It is frequently used in cancer research, aging studies, stem cell biology, and studies of telomere biology.

Advantages include high spatial resolution, compatibility with other FISH methods, and the ability to measure multiple loci per cell. Limitations include reliance on hybridization efficiency and imaging conditions, potential variability between laboratories, and the need for calibration to convert intensity to absolute length.