Commentary: The reliability of telomere length measurements (original) (raw)
Related papers
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.
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.
Evaluating minimally invasive sample collection methods for telomere length measurement
Objectives: Telomere length (TL) is a biomarker of aging and age-related decline. Although venous blood is considered the " gold standard " for TL measurement, its collection is often not feasible or desired in nonclinical settings. Saliva and dried blood spots (DBS) have been used as alternatives when venipuncture cannot be performed. However, it is not known whether these sample types yield TL measurements comparable to those obtained from venous blood. We sought to determine whether different samples from the same individual yield comparable TL measurements. Methods: We extracted DNA from matched buffy coat, saliva (Oragene and Oasis), and DBS (venous and capillary) samples from 40 women aged 18-77 years. We used the monochrome multiplex qPCR (MMQPCR) assay to measure TL in all sample types for each participant and applied quality control measures to retain only high-quality samples for analysis. We then compared TL from buffy coat and saliva to examine how these measurements differ and to test if TL is correlated across sample types. Results: TL differed significantly across buffy coat, Oragene saliva, and Oasis saliva samples. TL from buffy coat and Oragene saliva was moderately correlated (q 5 0.48, P 5 .002) and the most similar in size. Oasis saliva TL was not correlated with buffy coat or Oragene saliva TL, and was the shortest. DBS DNA yields were inadequate for TL measurement using the MMQPCR assay. Conclusions: Using a matched dataset we demonstrate that sample type significantly influences the TL measurement obtained using the MMQPCR assay.
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.
Leukocyte telomere length variation due to DNA extraction method
Background: Telomere length is indicative of biological age. Shorter telomeres have been associated with several disease and health states. There are inconsistencies throughout the literature amongst relative telomere length measured by quantitative PCR (qPCR) and different extraction methods or kits used. We quantified whole-blood leukocyte telomere length using the telomere to single copy gene (T/S) ratio by qPCR in 20 young (18-25 yrs) men after extracting DNA using three common extraction methods: Lahiri and Nurnberger (high salt) method, PureLink Genomic DNA Mini kit (Life Technologies) and QiaAmp DNA Mini kit (Qiagen). Telomere length differences of DNA extracted from the three extraction methods was assessed by one-way analysis of variance (ANOVA). Results: DNA purity differed between extraction methods used (P = 0.01). Telomere length was impacted by the DNA extraction method used (P = 0.01). Telomeres extracted using the Lahiri and Nurnberger method (mean T/S ratio: 2.43, range: 1.57 – 3.02) and PureLink Genomic DNA Mini Kit (mean T/S ratio: 2.57, range: 2.24 – 2.80) did not differ (P = 0.13). Likewise, QiaAmp and Purelink-extracted telomeres were not statistically different (P = 0.14). The Lahiri-extracted telomeres, however, were significantly shorter than those extracted using the QiaAmp DNA Mini Kit (mean T/S ratio: 2.71, range: 2.32 – 3.02; P = 0.003). DNA purity was associated with telomere length. Conclusion: There are discrepancies between the length of leukocyte telomeres extracted from the same individuals according to the DNA extraction method used. DNA purity could be responsible for the discrepancy in telomere length but this will require validation studies. We recommend using the same DNA extraction kit when quantifying leukocyte telomere length by qPCR or when comparing different cohorts to avoid erroneous associations between telomere length and traits of interest.
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 ...
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,
Nature Aging, 2022
Leukocyte telomere length (LTL) is a proposed marker of biological age. Here we report the measurement and initial characterization of LTL in 474,074 participants in UK Biobank. We confirm that older age and male sex associate with shorter LTL, with women on average ~7 years younger in "biological age" than men. Compared to white Europeans, LTL is markedly longer in African and Chinese ancestries. Older paternal 5 age at birth is associated with longer individual LTL. Higher white cell count is associated with shorter LTL, but proportions of white cell subtypes show weaker associations. Age, ethnicity, sex and white cell count explain ~5.5% of LTL variance. Using paired samples from 1,351 participants taken ~5 years apart, we estimate the within-individual variability in LTL and provide a correction factor for this. This resource provides opportunities to investigate determinants and biomedical consequences of variation in LTL.