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An Optimised Step-by-Step Protocol for Measuring Relative Telomere Length.

ABSTRACT: Telomeres represent the nucleotide repeat sequences at the ends of chromosomes and are essential for chromosome stability. They can shorten at each round of DNA replication mainly because of incomplete DNA synthesis of the lagging strand. Reduced relative telomere length is associated with aging and a range of disease states. Different methods such as terminal restriction fragment analysis, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization are available to measure telomere length; however, the qPCR-based method is commonly used for large population-based studies. There are multiple variations across qPCR-based methods, including the choice of the single-copy gene, primer sequences, reagents, and data analysis methods in the different reported studies so far. Here, we provide a detailed step-by-step protocol that we have optimized and successfully tested in the hands of other users. This protocol will help researchers interested in measuring relative telomere lengths in cells or across larger clinical cohort/study samples to determine associations of telomere length with health and disease.

SUBMITTER: Joglekar MV

PROVIDER: S-EPMC7359711 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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An Optimised Step-by-Step Protocol for Measuring Relative Telomere Length.

Joglekar Mugdha V MV Satoor Sarang N SN Wong Wilson K M WKM Cheng Feifei F Ma Ronald C W RCW Hardikar Anandwardhan A AA

Methods and protocols 20200403 2

Telomeres represent the nucleotide repeat sequences at the ends of chromosomes and are essential for chromosome stability. They can shorten at each round of DNA replication mainly because of incomplete DNA synthesis of the lagging strand. Reduced relative telomere length is associated with aging and a range of disease states. Different methods such as terminal restriction fragment analysis, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization are available to measure telomere ...[more]

PMID: 32260112

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Project description:(1) Background: Telomeres are repetitive DNA sequences located at the extremities of chromosomes that maintain genetic stability. Telomere biology is relevant to several human disorders and diseases, specifically cardiovascular disease. To better understand the link between cardiovascular disease and telomere length, we studied the effect of relative telomere length (RTL) on cardiovascular risk factors in a large population-based sample. (2) Methods: RTL was measured by a real-time quantitative polymerase chain reaction in subjects of the population-based Gutenberg Health Study (n = 4944). We then performed an association study of RTL with known cardiovascular risk factors of smoking status as well as systolic and diastolic blood pressure, body mass index (BMI), LDL cholesterol, HDL cholesterol, and triglycerides. (3) Results: A significant correlation was shown for RTL, with age as a quality control in our study (effect = -0.004, p = 3.2 × 10-47). Analysis of the relation between RTL and cardiovascular risk factors showed a significant association of RTL in patients who were current smokers (effect = -0.016, p = 0.048). No significant associations with RTL were seen for cardiovascular risk factors of LDL cholesterol (p = 0.127), HDL cholesterol (p = 0.713), triglycerides (p = 0.359), smoking (p = 0.328), diastolic blood pressure (p = 0.615), systolic blood pressure (p = 0.949), or BMI (p = 0.903). In a subsequent analysis, we calculated the tertiles of RTL. No significant difference across RTL tertiles was detectable for BMI, blood pressure, lipid levels, or smoking status. Finally, we studied the association of RTL and cardiovascular risk factors stratified by tertiles of age. We found a significant association of RTL and LDL cholesterol in the oldest tertile of age (effect = 0.0004, p = 0.006). (4) Conclusions: We determined the association of relative telomere length and cardiovascular risk factors in a population setting. An association of telomere length with age, current smoking status, as well as with LDL cholesterol in the oldest tertile of age was found, whereas no associations were observed between telomere length and triglycerides, HDL cholesterol, blood pressure, or BMI.

Dataset Information

An Optimised Step-by-Step Protocol for Measuring Relative Telomere Length.

Publications

An Optimised Step-by-Step Protocol for Measuring Relative Telomere Length.

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