Telomere length measurements using digital fluorescence microscopy.
BACKGROUND
The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH).


METHODS
Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user.


RESULTS
Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained.


CONCLUSIONS
We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.
Poon
S S
SS
Terry Fox Laboratory for Hematology/Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada.
Martens
U M
UM
Ward
R K
RK
Lansdorp
P M
PM
D000465:Algorithms; D002138:Calibration; D002232:Carbocyanines; D004247:DNA / Q000032:analysis*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007211:Indoles; D007621:Karyotyping; D008214:Lymphocytes / Q000648:ultrastructure; D008297:Male; D008677:Metaphase; D008856:Microscopy, Fluorescence / Q000379:methods*; D010316:Particle Size; D012091:Repetitive Sequences, Nucleic Acid; D012815:Signal Processing, Computer-Assisted; D016615:Telomere / Q000737:chemistry* / Q000648:ultrastructure
D002232:Carbocyanines; D007211:Indoles; C089187:cyanine dye 3; C007293:DAPI; D004247:DNA