Telomere Length Measurement by FISH

Abstract

Telomere length influences numerous cellular processes such as senescence, carcinogenesis, and aging. Quantitative FISH (Q-FISH) is a comprehensive method that allows measuring of individual chromosome telomere length in single cell with the resolution of 200 base pairs. The method is based on the use of a peptide nucleic acid (PNA) telomere oligonucleotide probe and appropriate digital image software for capture and quantification of fluorescence signals. The length of telomere is directly related to its integrated fluorescence intensity, as PNA probes are assumed to hybridize quantitatively to telomeric repeats. For the accuracy of Q-FISH measurement, it is important to use adequate internal controls such as fluorescence beads of defined size to avoid imprecisions due to lamp intensity variations. Fluorescence intensity of beads is then used to correct fluorescence intensities of telomere signals. Telomeric/centromeric ratio or calibration relative to cultured cell of known telomere length allows fluorescence intensity values to be converted into units of DNA length (RTLU). However, this step is not essential as fluorescence measurements in arbitrary units (TFU) will yield accurate results given that internal control (i.e., fluorescence beads) is used properly.