TELOMERE ANALYSIS BY FLUORESCENCE IN-SITU HYBRIDIZATION AND FLOW-CYTOMETRY

Determination of telomere length is traditionally performed by Souther n blotting and densitometry, giving a mean telomere restriction fragme nt (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calcula te telomere length, a method called quantitative (Q)-FISH, We here pre sent a quantitative flow cytometric approach, Q-FISHFCM, for evaluatio n of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)(3) probe and DMA staining with propidium iodide, A simple and rapid protocol with results within 30 h was developed giving high rep roducibility, One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for poten tial differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, bloo d, lymph nodes and tonsils. A significant correlation was found betwee n Southern blotting and Q-FISHFCM telomere length values (P = 0.002), The mean sub-telomeric DNA length of the tested cell lines and clinica l samples was estimated to be 3.2 kbp, With the Q-FISHFCM method the f luorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.