Letter to the Editor Telomere length measurement validity: the coefficient of variation is invalid and cannot be used to compare quantitative polymerase chain reaction and Southern blot telomere length measurement techniques (original) (raw)
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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,
Scientific Reports, 2016
Measurement of telomere length is widely used in epidemiologic studies. Insufficient standardization of the measurements processes has, however, complicated the comparison of results between studies. We aimed to investigate whether DNA extraction methods have an influence on measured values of relative telomere length (RTL) and whether this has consequences for epidemiological studies. We performed four experiments with RTL measurement in quadruplicate by qPCR using DNA extracted with different methods: 1) a standardized validation experiment including three extraction methods (magnetic-particle-method EZ1, salting-out-method INV, phenol-chloroform-isoamyl-alcohol PCI) each in the same 20 samples demonstrated pronounced differences in RTL with lowest values with EZ1 followed by INV and PCI-isolated DNA; 2) a comparison of 307 samples from an epidemiological study showing EZ1-measurements 40% lower than INV-measurements; 3) a matching-approach of two similar non-diseased control groups including 143 pairs of subjects revealed significantly shorter RTL in EZ1 than INV-extracted DNA (0.844 ± 0.157 vs. 1.357 ± 0.242); 4) an association analysis of RTL with prevalent cardiovascular disease detected a stronger association with INV than with EZ1-extracted DNA. In summary, DNA extraction methods have a pronounced influence on the measured RTL-values. This might result in spurious or lost associations in epidemiological studies under certain circumstances. Telomeres and telomerase discovered several decades ago are considered a "protection machinery" of our genome 1-4. Telomeres have been under intensive investigation due to the hypothesis that they may be responsible for aging on the cellular level and affect lifespan 5,6. Many independent cross-sectional studies postulate an association of short telomere length (TL) with higher risk for various diseases such as cancer and atherosclerosis including its comorbidities. Additionally, shorter TL has been related to mortality 7 and a variety of diseases 8-17. Recently, data from prospective cohort studies including TL measurement at two different time points became available. From these studies evidence is accumulating that TL dynamics are not a one-way road with shortening over time 18-20. Lengthening of telomeres can occur as well, which was observed in a large proportion (44% of 4,576 individuals) of the general population 20. Altogether, a sinusoidal behavior of telomere length over time can be observed which decreases on average with age. Telomere length can be measured by different methods. Widely used techniques are the absolute measurement of telomere length with restriction fragments analysis by Southern blot 21,22 and the relative measurement by real-time quantitative polymerase chain reaction (qPCR) 23. The latter is a frequently used method in epidemiological studies since much less DNA is required and it is less laborious allowing a high-throughput approach. Therefore, for all of our studies, we applied the well-established and as automated as possible high-throughput qPCR method for measurement of relative telomere length (RTL) with a high level of standardization to ensure reliable and high quality data for epidemiological studies. We generally perform RTL measurements in quadruplicate to maximize accuracy.
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.
2019
Objectives: Telomeres are the protective caps of chromosomes. They shorten with cell replication, age, and possibly environmental stimuli (e.g., infection and stress). Short telomere length (TL) predicts subsequent worse health. Although venous whole blood (VWB) is most commonly used for TL measurement, other, more minimally-invasive, sampling techniques are becoming increasingly common due to their field-friendliness, allowing for feasible measurement in low-resource contexts. We conducted validation work for measuring TL in dried blood spots (DBS) and incorporated our results into a meta-analysis evaluating minimally-invasive sampling techniques to measure TL.Methods: We isolated DNA extracts from DBS using a modified extraction protocol and tested how they endured different shipping conditions and long-term cryostorage. We then included our in-house DBS TL validation statistics (correlation values with VWB TL and age) in a series of meta-analyses of results from 24 other studies ...
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.
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.
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;
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.
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.