Potential Risks in the Paradigm of Basic to Translational Research: A Critical Evaluation of qPCR Telomere Size 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,
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.
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.
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.
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.
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...
Guest Editorial Improving Precision in Investigating Aging: Why Telomeres Can Cause Problems
A host of recent publications has highlighted a growing number of discrepancies between small-scale laboratory-based studies and larger clinical and epidemiological studies, using telomere length as a bio-aging marker for physical, sociological, and psychological parameters in their respective cohorts. These discrepancies may be rooted in differing telomere length measurement methods and their application. This leads to the question of just how robust telomere length is as a biomarker of aging and whether measurement of CDKN2A levels offers a better alternative. The latter has already provided reproducible data from a small number of clinical studies and in one proven better than telomere length determination in predicting organ function. It seems prudent to address the use of these markers, alone or in combination, in multicentre double-blinded studies, using standardized methodologies and reagents, in order to identify the most appropriate marker and method for investigating bio-aging.
Choice of Isolation method has a significant impact on average murine Telomere Length estimates
Background Telomere Length (TL) and integrity is significantly associated with age-related disease, multiple genetic and environmental factors. We observe mouse genomic DNA (gDNA) isolation methods have a significant impact on average TL estimates. The canonical qPCR method does not measure TL directly but via the ratio of telomere repeats to a single copy gene (SCG) generating an TS ratio. We use an mmqPCR method which multiplexes the PCR and enables quantification of the target and the single copy gene within the same qPCR reaction. Results We demonstrate TL measurements, from murine gDNA, isolated via Spin Columns (SC) and Magnetic Beads (MB), generate significantly smaller T/S ratios compared to gDNA isolated via traditional phenol/chloroform methods. The former methods may impede correct TL estimation by producing non representative fragment sets and reducing qPCR efficacy. Conclusions This work highlights discrepancies in TL measurements due to different extraction techniques....