Influence of DNA extraction methods on relative telomere length measurements and its impact on epidemiological studies (original) (raw)
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Data from Telomere Length Varies By DNA Extraction Method: Implications for Epidemiologic Research
2023
Background: Both shorter and longer telomeres in peripheral blood leukocyte (PBL) DNA have been associated with cancer risk. However, associations remain inconsistent across studies of the same cancer type. This study compares DNA preparation methods to determine telomere length from patients with colorectal cancer. Methods: We examined PBL relative telomere length (RTL) measured by quantitative PCR (qPCR) in 1,033 patients with colorectal cancer and 2,952 healthy controls. DNA was extracted with phenol/chloroform, PureGene, or QIAamp. Results: We observed differences in RTL depending on DNA extraction method (P < 0.001). Phenol/ chloroform-extracted DNA had a mean RTL (T/S ratio) of 0.78 (range 0.01-6.54) compared with PureGeneextracted DNA (mean RTL of 0.75; range 0.00-12.33). DNA extracted by QIAamp yielded a mean RTL of 0.38 (range 0.02-3.69). We subsequently compared RTL measured by qPCR from an independent set of 20 colorectal cancer cases and 24 normal controls in PBL DNA extracted by each of the three extraction methods. The range of RTL measured by qPCR from QIAamp-extracted DNA (0.17-0.58) was less than from either PureGene or phenol/chloroform (ranges, 0.04-2.67 and 0.32-2.81, respectively). Conclusions: RTL measured by qPCR from QIAamp-extracted DNA was less than from either PureGene or phenol/chloroform (P < 0.001). Impact: Differences in DNA extraction method may contribute to the discrepancies between studies seeking to find an association between the risk of cancer or other diseases and RTL. Cancer Epidemiol Biomarkers Prev; 22(11); 2047-54. Ó2013 AACR.
Telomere Length Varies By DNA Extraction Method: Implications for Epidemiologic Research
Cancer Epidemiology Biomarkers & Prevention, 2013
Background: Both shorter and longer telomeres in peripheral blood leukocyte (PBL) DNA have been associated with cancer risk. However, associations remain inconsistent across studies of the same cancer type. This study compares DNA preparation methods to determine telomere length from patients with colorectal cancer. Methods: We examined PBL relative telomere length (RTL) measured by quantitative PCR (qPCR) in 1,033 patients with colorectal cancer and 2,952 healthy controls. DNA was extracted with phenol/chloroform, PureGene, or QIAamp. Results: We observed differences in RTL depending on DNA extraction method (P < 0.001). Phenol/ chloroform-extracted DNA had a mean RTL (T/S ratio) of 0.78 (range 0.01-6.54) compared with PureGeneextracted DNA (mean RTL of 0.75; range 0.00-12.33). DNA extracted by QIAamp yielded a mean RTL of 0.38 (range 0.02-3.69). We subsequently compared RTL measured by qPCR from an independent set of 20 colorectal cancer cases and 24 normal controls in PBL DNA extracted by each of the three extraction methods. The range of RTL measured by qPCR from QIAamp-extracted DNA (0.17-0.58) was less than from either PureGene or phenol/chloroform (ranges, 0.04-2.67 and 0.32-2.81, respectively). Conclusions: RTL measured by qPCR from QIAamp-extracted DNA was less than from either PureGene or phenol/chloroform (P < 0.001). Impact: Differences in DNA extraction method may contribute to the discrepancies between studies seeking to find an association between the risk of cancer or other diseases and RTL. Cancer Epidemiol Biomarkers Prev; 22(11); 2047-54. Ó2013 AACR.
Reproducibility of telomere length assessment: an international collaborative study
International Journal of Epidemiology, 2014
Background: Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories. Methods: We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intra-and inter-batch variation between laboratories and techniques. Results: Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63-0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However,
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.
American Journal of Human Biology, 2011
Objectives: Telomeres, repetitive DNA sequences found at the ends of chromosomes, shorten with age in proliferating human tissues and are implicated in senescence. Previous studies suggest that shorter telomeres impair immune and cardiovascular function and result in increased mortality. Although few, prior studies have documented ethnic/population differences in human telomere lengths. The nature and cause(s) of these population differences remain poorly understood.
Association between telomere length in blood and mortality in people aged 60 years or older
2003
During normal ageing, the gradual loss of telomeric DNA in dividing somatic cells can contribute to replicative senescence, apoptosis, or neoplastic transformation. In the genetic disorder dyskeratosis congenita, telomere shortening is accelerated, and patients have premature onset of many age-related diseases and early death. We aimed to assess an association between telomere length and mortality in 143 normal unrelated individuals over the age of 60 years. Those with shorter telomeres in blood DNA had poorer survival, attributable in part to a 3·18-fold higher mortality rate from heart disease (95% CI 1 . 36-7·45, p=0·0079), and an 8·54-fold higher mortality rate from infectious disease (1·52-47·9, p=0·015). These results lend support to the hypothesis that telomere shortening in human beings contributes to mortality in many age-related diseases.
Genetics, 2015
The Kaiser Permanente Research Program on Genes, Environment, and Health (RPGEH) Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort includes DNA specimens extracted from saliva samples of 110,266 individuals. Because of its relationship to aging, telomere length measurement was considered an important biomarker to develop on these subjects. To assay relative telomere length (TL) on this large cohort over a short time period, we created a novel high throughput robotic system for TL analysis and informatics. Samples were run in triplicate, along with control samples, in a randomized design. As part of quality control, we determined the within-sample variability and employed thresholds for the elimination of outlying measurements. Of 106,902 samples assayed, 105,539 (98.7%) passed all quality control (QC) measures. As expected, TL in general showed a decline with age and a sex difference. While telomeres showed a negative correlation with age up to 75 years, in those older than 75 years, age positively correlated with longer telomeres, indicative of an association of longer telomeres with more years of survival in those older than 75. Furthermore, while females in general had longer telomeres than males, this difference was significant only for those older than age 50. An additional novel finding was that the variance of TL between individuals increased with age. This study establishes reliable assay and analysis methodologies for measurement of TL in large, population-based human studies. The GERA cohort represents the largest currently available such resource, linked to comprehensive electronic health and genotype data for analysis. KEYWORDS relative telomere length; GERA cohort; saliva DNA; robotic assay; quantitative PCR T ELOMERES are the protective DNA-protein complexes that cap the ends of eukaryotic chromosomes and are required for genome stability. The essential telomeric DNA consists of a tract of a tandemly repeated short sequence specified and maintained by the highly regulated reverse transcriptase action of the cellular enzyme telomerase. Telomeric DNA is susceptible to natural terminal erosion through a variety of processes including the end replication problem of linear chromosomal DNA, which causes telomeres to get shorter each time a somatic cell divides (Olovnikov 1973;
The Journal of Applied Laboratory Medicine: An AACC Publication
Background: Average telomere length in whole blood has become a biomarker of aging, disease, and mortality risk across a broad range of clinical conditions. The most common method of telomere length measurement for large patient sample sets is based on quantitative PCR (qPCR). For laboratory-developed tests to be performed on clinical samples, they must undergo a rigorous analytical validation, currently regulated under CLIA. Methods: Whole blood samples from 40 donors were used in the analytical validation of methods for relative average telomere length (rATL) measurement. Three technical replicate DNA samples were extracted from each whole blood sample and placed in three independent wells on a sample plate. Each of these sample plates was assayed 12 times during the validation process. The study was conducted over a 20-day period, once in the morning and once in the evening, using 3 different operators. Results: Our process of rATL measurement beginning with DNA extraction followed by qPCR-based assay resulted in repeatability and reproducibility CV of <5% and amplification efficiencies near 100%. The validated assay was used to establish a reference interval derived from 2 cohorts of individuals: (a) San Francisco Bay area (n = 504) and (b) a US cross-sectional, demographic population (n = 357). Conclusions: We present advances in the establishment of a highly reproducible analytically validated process for determining rATLs in a CLIA laboratory environment. IMPACT STATEMENT Leukocyte telomere length is emerging as a biomarker for age-related disease risk. The challenge of comparing telomere length analyses across laboratories includes reconciling different methodologies, varied standardization, and high variability. Adoption of stringent controls and performance characteristics are central to pursuing clinical indications associated with small dynamic ranges of telomere lengths. We present the rigorous CLIA-inspired analytical validation of a relative average telomere length (rATL) measurement process, which we used to establish a normal reference interval. Given the growing number of associations between leukocyte telomere length and disease risk, particularly cardiac, increased consistency within and between assays benefits affected individuals.
Commentary: The reliability of telomere length measurements
International Journal of Epidemiology, 2015
The importance of telomere biology in human disease is increasingly recognized and, in parallel, use of telomere length (TL) measures is proliferating in epidemiological and clinical studies. Such studies measure leukocyte TL (LTL) using several methodological approaches. Shorter LTL is associated with atherosclerosis 1 and all-cause mortality. 2 Given the increasingly recognized role of TL in human ageing and its related diseases, it is essential to know more about the reliability and validity of TL measurement methods, their comparability and which method is optimal for a specific epidemiological/clinical setting.