Evaluation of Laboratory Methods to Measure Telomere Length in Cancer (original) (raw)
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The Journal of Pathology, 2003
Loss of telomere repeat sequences occurs after each cell division and telomere shortening has been implicated in cellular senescence. The measurement of telomere length might therefore assess the lifespan of a cell. The aim of this study was to set up and validate a technique enabling the assessment of telomere length on tissue sections. Quantitative fluorescence in situ hybridization (Q-FISH) with telomeric probes was performed on smears and sections from cell preparations or human tissues. The mean fluorescence intensity of telomere spots (FI/spot) was automatically quantified by image analysis. Telomeric restriction fragment (TRF) length was assessed by Southern blotting. There was a positive significant correlation between telomere length, as assessed by Q-FISH, and TRF length determined by Southern blotting in corresponding samples (p < 0.01, r = 0.6 for tissue and p < 0.01, r = 0.79 for cells). FI/spot was higher on smears than on sections, but pairwise comparison showed a significant correlation both for cells and for tissues (r = 0.77, p < 0.001 for cells and p ≤ 0.01, r = 0.64 for tissue). Finally, since telomere length is expected to shorten with age, FI/spot was assessed in liver samples according to the age of patients: a negative correlation was demonstrated (r = 0.76, p < 0.01). Inter-assay variation was 7% for Q-FISH performed on tissue sections and 12% on touch preparations. This study shows that Q-FISH can be performed with confidence on fixed frozen tissue sections in order to assess telomere length. It is an easy, accurate, and reproducible in situ method for assessing telomeres in the context of cell type and tissue architecture.
Cytometry, 2002
Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to ...
PLoS ONE, 2014
Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R 2 = 0.60; p,0.0001) and patients (R 2 = 0.51; p,0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R 2 = 0.35; p,0.0001) and low correlation for patients (R 2 = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R 2 = 0.33; p,0.0001) and did not correlate in the comparison of patients' samples (R 2 = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.867.1%) and qPCR (9.567.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.667.6% vs. 16619.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes.
Biological Procedures Online, 2020
Background A large number of studies have suggested a correlation between the status of telomeres and disease risk. High-throughput quantitative fluorescence in situ hybridization (HT Q-FISH) is a highly accurate telomere measurement technique that can be applied to the study of large cell populations. Here we describe the analytical performance testing and validation of Telomere Analysis Technology (TAT®), a laboratory-developed HT Q-FISH-based methodology that includes HT imaging and software workflows that provide a highly detailed view of telomere populations. Methods TAT was developed for the analysis of telomeres in peripheral blood mononuclear cells (PBMCs). TAT was compared with Terminal Restriction Fragment (TRF) length analysis, and tested for accuracy, precision, limits of detection (LOD) and specificity, reportable range and reference range. Results Using 6 different lymphocyte cell lines, we found a high correlation between TAT and TRF for telomere length (R2 ≥ 0.99). T...
Measurement of telomere length: a new assay using QuantiGene chemistry on a Luminex platform
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 2014
Telomeres are tandem repeats of sequences present at the end of the chromosomes that maintain chromosomal integrity. After repeated cell division, telomeres shorten to a critical level, triggering replicative senescence or apoptosis, which is a key determinant of cellular aging. Short telomeres also contribute to genome instability and are a hallmark of many cancers. There are several methods for estimating telomere length (TL) from extracted DNA samples. Southern blot is accurate but requires a large quantity of DNA and is expensive. qPCR is cost-effective and requires a small quantity of DNA and is therefore widely used for large-scale epidemiologic studies; however, it typically requires triplicates. We describe a novel multiplexed probe-based non-PCR method for TL measurement. A small amount of DNA (∼50 ng) is hybridized to telomere repeat sequence-specific probes (T) and a reference single gene probes (R). T and R signals are detected from a single reaction well containing the ...
Measurement of telomere DNA content by dot blot analysis
Nucleic Acids Research, 2011
Telomeres play a central role in human cancer, cardiovascular aging and possibly longevity. However, present methods to measure telomere length are fraught with shortcomings that limit their use. Here, we describe a novel method to measure the relative telomere DNA content by dot blot analysis. In each dot, the DNA content is measured by a DNA stain (Dx) and the telomeric DNA content is measured with a telomeric probe (T). The T normalized for Dx (T/Dx) of each dot is a measure of telomere content. The method requires $20 ng of DNA per assay. Moreover, the T/Dx data are highly correlated linearly with mean telomere lengths derived from Southern blots of the terminal restriction fragments (r > 0.96, P < 0.0001). The method is also simple to use, has a relatively low interassay coefficient of variation (<6%), retains its precision in moderately degraded DNA and can be forged for high throughput analysis. The method might help researchers and clinicians alike in understanding risks for and extent of human diseases.
Anticancer research
The end of eukaryotic chromosomes terminates with nucleoprotein structures called telomeres. They insure several functions including capping the end of the chromosomes, ensuring their stability and protecting them from end-to-end fusion and preventing the activation of the DNA damage checkpoints. A flow-FISH methodology, i.e. quantitative fluorescence in situ hybridization (Q-FISH) in combination with flow cytometry, has been developed in our laboratory in order to estimate telomere length in three human cancer cell lines: K-562 (chronic myelogenous leukaemia), IM-9 (multiple myeloma) and 1301 (T cell lymphoblastic leukaemia). Telomeres were visualised after hybridisation with FITC-labelled PNA (Peptide Nucleic Acid) probes. We evaluated the most critical steps of the flow-FISH protocol to ensure reproducibility. Different methodological set ups were compared. Three fixation procedures (ethanol 80%, methanol 80% and formaldehyde 4%) were tested besides different fixation times (15 m...
Measurement of Telomeric DNA Content in Human Tissues
BioTechniques, 1997
Telomeres, nucleoprotein complexes at the ends of eukaryotic chromosomes, are 10-12 kbp in length in somatic cells, but as small as 1-2 kbp in rapidly growing cancer cells. Southern blot analysis is currently the standard method for the measurement of telomere length. However, accurate determinations are not possible when DNA is broken or scant. To avoid these problems, a slot blot assay that quantitates the relative content, instead of length, of telomere DNA was developed. The relative contents of telomere DNA determined by this slot blot assay were directly proportional to the relative lengths of telomere DNA determined in parallel by Southern blot analysis. Relative telomere DNA content could be measured in samples containing as little as 15 ng of total DNA. Relative telomere DNA content, but not length, also was unaffected by breakage of DNA into fragments 1 kbp or less in length.
2018
Aim: Telomere length (TL) measurement by quantitative polymerase chain reaction (PCR) has been widely accepted, but limited information regarding its validation with a gold-standard technique is available. Materials & methods: We measured TL by Southern blot and monochrome multiplex quantitative PCR (MMqPCR) and validated the results of TL in leukocytes of 94 participants with mean age 43.2 years, BMI 19-41 (mean 27.8 ± 4.3) kg/m 2. Results: A significant positive correlation was observed between TL measured by MMqPCR and Southern blot assay (correlation coefficient r = +0.896, p < 0.0001). The inter-and intra-assay CVs of the MMqPCR assay were 5.3 and 4.07%, respectively. Conclusion: We observed that experimental discrepancies have an impact on TL analysis and there is a need to improve the optimum conditions.