Telomere length assessment in tissue sections by quantitative FISH: image analysis algorithms.
BACKGROUND
Telomeres are tandem repeated DNA sequences at the ends of every chromosome, which cap, stabilize, and prevent chromosome fusions and instability. Telomere regulation is an important mechanism in cellular proliferation and senescence in normal diploid and neoplastic cells. Quantitative methods to assess telomere lengths are essential to understanding how telomere dynamics play a role in these processes.


METHODS
Telomere lengths have been conventionally measured using terminal restriction fragment (TRF), quantitative fluorescence in situ hybridization (QFISH), and flow FISH. In this study, we have applied QFISH to measure average telomere lengths in cultured cells and human tissues of the GI tract. Importantly, this method can be used to analyze telomere lengths in sections using confocal microscopy. We describe and compare three image analysis algorithms: a simple pixel histogram calculation of background corrected fluorescence, a telomere spot-finding method, and a background curve subtraction algorithm.


RESULTS
Using normal human diploid fibroblasts (NHDF) either dropped on slides or sectioned after agar embedding, similar telomere length shortening is evident with increasing population doubling levels (PDLs), using peptide nucleic acid (PNA) and an N3'-P5'-phosphoamidate (PA) oligonucleotide probe for all three methods. Validation of these in situ telomere quantification methods showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. Telomere length reductions can also be demonstrated in tissue sections from histologically normal mucosa from patients with chronic ulcerative colitis (with dysplasia or cancer elsewhere in the colon), in colon adenomas, and in mucosal biopsies from patients with Barrett's esophagus. Both on slides and in tissue sections, the telomere spot-finding method has the greatest variability, while intra- and inter-biopsy variability in telomere length assessment using the other methods is relatively low.


CONCLUSIONS
Accurate and reproducible telomere length measurements can be made in tissue sections using QFISH and confocal microscopy. The simplest methods proved the most reliable and make these methods readily accessible to many laboratories. The use of these methods will enhance the ability to measure telomere lengths in tissue samples and aid in the understanding of the role of telomere length in aging and disease.
O'Sullivan
Jacintha N
JN
Department of Pathology, University of Washington, Seattle, Washington 98195-7705, USA.
Finley
Jennifer C
JC
Risques
Rosa-ana
RA
Shen
Wen-Tang
WT
Gollahon
Katherine A
KA
Moskovitz
Alexander H
AH
Gryaznov
Sergei
S
Harley
Calvin B
CB
Rabinovitch
Peter S
PS
D000465:Algorithms; D001471:Barrett Esophagus / Q000473:pathology; D001706:Biopsy; D002460:Cell Line; D016922:Cellular Senescence; D002503:Centromere / Q000235:genetics / Q000378:metabolism; D003093:Colitis, Ulcerative / Q000473:pathology; D003106:Colon / Q000473:pathology; D005347:Fibroblasts; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal; D015345:Oligonucleotide Probes / Q000032:analysis / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000032:analysis / Q000235:genetics; D016615:Telomere / Q000235:genetics / Q000378:metabolism*
D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids