Telomere Length Calibration from qPCR Measurement: Limitations of Current Method

Telomere length (TL) comparisons from different methods are challenging due to differences in laboratory techniques and data configuration. This study aimed to assess the validity of converting the quantitative polymerase chain reaction (qPCR) telomere/single copy gene (T/S) ratio to TL in kilobases (kb). We developed a linear regression equation to predict TL from qPCR T/S using flow cytometry with fluorescence in situ hybridization (flow FISH) TL data from 181 healthy donors (age range = 19–53) from the National Marrow Donor Program (NMDP) biorepository. TL measurements by qPCR and flow FISH were modestly correlated (R2 = 0.56, p < 0.0001). In Bland-Altman analyses, individuals with the shortest (≤10th percentile) or longest (≥90th) flow FISH TL had an over- or under-estimated qPCR TL (bias = 0.89 and −0.77 kb, respectively). Comparisons of calculated TL from the NMDP samples and 1810 age- and sex-matched individuals from the National Health and Nutrition Examination Survey showed significant differences (median = 7.1 versus 5.8 kb, respectively, p < 0.0001). Differences in annual TL attrition were also noted (31 versus 13 bp/year, respectively, p = 0.02). Our results demonstrate that TL calculated in kb from qPCR T/S may yield biased estimates for individuals with the shortest or longest TL, those often of high clinical interest. We also showed that calculated TL in kb from qPCR data are not comparable across populations and therefore are not necessarily useful.

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

Methods and Protocols ◽

10.3390/mps3020027 ◽

2020 ◽

Vol 3 (2) ◽

pp. 27 ◽

Cited By ~ 3

Author(s):

Mugdha V. Joglekar ◽

Sarang N. Satoor ◽

Wilson K.M. Wong ◽

Feifei Cheng ◽

Ronald C.W. Ma ◽

...

Keyword(s):

Telomere Length ◽

Large Population ◽

Single Copy ◽

Population Based ◽

Fragment Analysis ◽

Real Time Quantitative Pcr ◽

Disease States ◽

Relative Telomere Length ◽

Health And Disease

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.

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The relationships of lifetime physical activity and diet with salivary cell telomere length in current ultra-endurance exercisers

Nutrition and Healthy Aging ◽

10.3233/nha-200090 ◽

2021 ◽

pp. 1-11

Author(s):

Karen Birkenhead ◽

Anna Kuballa ◽

Geoff P. Lovell ◽

Susan I. Barr ◽

Colin Solomon

Keyword(s):

Physical Activity ◽

Telomere Length ◽

Endurance Exercise ◽

Dna Sequences ◽

Quantitative Polymerase Chain Reaction ◽

Food Category ◽

Age Related ◽

Salivary Cell ◽

Ultra Endurance ◽

Age Range

BACKGROUND: Physical activity and a healthy diet may delay the aging process and ultra-endurance exercise is an extreme form of physical activity. Telomeres are protective DNA sequences located at the ends of eukaryotic chromosomes which shorten as we age. OBJECTIVE: The aim of this study was to investigate the relationships of lifetime physical activity and diet with salivary cell telomere length in current ultra-endurance exercisers (n = 49; %female = 37, age range 26–74 years). METHODS: Physical activity and dietary intake were measured using the Lifetime Physical Activity and Diet Questionnaire (LPADQ) and salivary cell telomere length was measured using quantitative polymerase chain reaction. RESULTS: In this group of current ultra-endurance exercisers there was no relationship between lifetime physical activity or diet (according to food category scores) and telomere length. In contrast to the expected age-related decrease in telomere length, there was no relationship between age and telomere length (95%confidence interval [CI]: –38.86, 14.54, p = 0.359) in this group of current ultra-endurance exercisers. CONCLUSIONS: The relationships of lifetime physical activity and diet with telomere length remain uncertain. It is possible that lifetime physical activity (including ultra-endurance exercise) and lifetime diet may independently, or in combination, contribute to a decrease in the rate of age-related telomere shortening in current ultra-endurance exercisers. ultra-endurance exercisers.

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Early maternal separation is not associated with changes in telomere length in domestic kittens (Felis catus)

PeerJ ◽

10.7717/peerj.11394 ◽

2021 ◽

Vol 9 ◽

pp. e11394

Author(s):

Mikel Delgado ◽

C.A. Tony Buffington ◽

Melissa Bain ◽

Dana L. Smith ◽

Karen Vernau

Keyword(s):

Telomere Length ◽

Mixed Model ◽

Maternal Separation ◽

Linear Mixed Model ◽

Quantitative Polymerase Chain Reaction ◽

Single Copy ◽

Felis Catus ◽

Telomere Shortening ◽

Orphan Status ◽

Animal Rescue

Objective Studies of multiple species have found that adverse early life experiences, including childhood trauma and maternal separation, can result in accelerated telomere shortening. The objective of this study was to determine if premature separation from the mother affected telomere length in domestic kittens (Felis catus). Subjects were 42 orphaned kittens and 10 mother-reared kittens from local animal rescue groups and shelters. DNA was extracted from whole blood collected from kittens at approximately 1 week and 2 months of age. Telomere length was assessed by qPCR (quantitative polymerase chain reaction) from a total of 86 samples and expressed as a ratio of telomere PCR relative to a single copy gene PCR (T/S). Results A generalized linear mixed model found there were no detectable differences in telomere length based on survival (F1, 76.2 = 3.35, p = 0.07), orphan status (F1, 56.5 = 0.44, p = 0.51), time point (F1, 43.5 = 0.19, p = 0.67), or the interaction between orphan status and time (F1, 43.5 = 0.86, p = 0.36). Although in other species telomere shortening is commonly associated with aging, even early in life, we did not find evidence for telomere shortening by two months of age. Our results suggest that the experience of early maternal separation in domestic cats who are subsequently hand-reared by humans does not accelerate telomere shortening compared to mother-reared kittens, at least in the first few months of life.

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Landscape of Granulocyte Telomere Shortening in Cytopenia in India

10.21203/rs.3.rs-36887/v1 ◽

2020 ◽

Author(s):

PARAMITA MANDAL ◽

Shyamali Dutta

Keyword(s):

Telomere Length ◽

Peptide Nucleic Acid ◽

Large Population ◽

Inverse Correlation ◽

Telomerase Activity ◽

Blood Leukocytes ◽

Telomere Shortening ◽

Healthy Donors ◽

Potential Use

Abstract Objectives: Telomere length and telomerase activity have been implicated in the control of cell proliferation and genomic stability. We recently introduced a novel technique for measurement of the average telomere length in cells using fluorescence in situ hybridization (FISH) with peptide nucleic acid (PNA) probes and flow cytometry (flow-FISH). Results: Using this technique, we have analyzed the telomere length kinetics in subpopulations of unseparated peripheral blood leukocytes (PBLs) in a large population of healthy donors as well as patients with cytopenia. We only found significantly shortened telomeres from patient with cytopenia compared to age-adjusted controls. Strikingly, the telomere length in granulocytes from cytopenia patients differed significantly from controls. Furthermore, an inverse correlation between of granulocyte telomere length and age was found among healthy controls. However, surprisingly, telomere length in cytopenia patients with hypocellular marrow was significantly shorter from patients with non-hypocellular marrow. These results support the concept of accelerated granulocyte telomere shortening in cytopenia patients in India and suggest a potential use of telomere-length measurements as a prognostic tool in this group of disorders as well.

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Telomere Length as a Biomarker for Race-Related Health Disparities

Genes ◽

10.3390/genes12010078 ◽

2021 ◽

Vol 12 (1) ◽

pp. 78

Author(s):

Vaithinathan Selvaraju ◽

Megan Phillips ◽

Anna Fouty ◽

Jeganathan Ramesh Babu ◽

Thangiah Geetha

Keyword(s):

Blood Pressure ◽

Telomere Length ◽

Diastolic Pressure ◽

Systolic Pressure ◽

Single Copy ◽

Normal Weight ◽

Length Ratio ◽

Hierarchical Regression ◽

Significant Difference ◽

The Usa

Disparities between the races have been well documented in health and disease in the USA. Recent studies show that telomere length, a marker of aging, is associated with obesity and obesity-related diseases, such as heart disease and diabetes. The current study aimed to evaluate the connection between telomere length ratio, blood pressure, and childhood obesity. The telomere length ratio was measured in 127 children from both European American (EA) and African American (AA) children, aged 6–10 years old. AA children had a significantly high relative telomere to the single copy gene (T/S) ratio compared to EA children. There was no significant difference in the T/S ratio between normal weight (NW) and overweight/obese (OW/OB) groups of either race. Blood pressure was significantly elevated in AA children with respect to EA children. Hierarchical regression analysis adjusted for race, gender, and age expressed a significant relationship between the T/S ratio and diastolic pressure. Low T/S ratio participants showed a significant increase in systolic pressure, while a high T/S ratio group showed an increase in diastolic pressure and heart rate of AA children. In conclusion, our findings show that AA children have high T/S ratio compared to EA children. The high T/S ratio is negatively associated with diastolic pressure.

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Association of Metabolically Healthy and Unhealthy Obesity Phenotypes with Oxidative Stress Parameters and Telomere Length in Healthy Young Adult Men. Analysis of the MAGNETIC Study

Antioxidants ◽

10.3390/antiox10010093 ◽

2021 ◽

Vol 10 (1) ◽

pp. 93

Author(s):

Mateusz Lejawa ◽

Kamila Osadnik ◽

Tadeusz Osadnik ◽

Natalia Pawlas

Keyword(s):

Oxidative Stress ◽

Telomere Length ◽

Quantitative Polymerase Chain Reaction ◽

Oxidative Stress Markers ◽

Metabolic Disturbances ◽

Total Oxidation ◽

Stress Markers ◽

Metabolic Dysregulation ◽

Total Antioxidant ◽

Metabolically Healthy

Obesity is a significant factor related to metabolic disturbances that can lead to metabolic syndrome (MetS). Metabolic dysregulation causes oxidative stress, which affects telomere structure. The current study aimed to evaluate the relationships between telomere length, oxidative stress and the metabolically healthy and unhealthy phenotypes in healthy young men. Ninety-eight participants were included in the study (49 healthy slim and 49 obese patients). Study participants were divided into three subgroups according to body mass index and metabolic health. Selected oxidative stress markers were measured in serum. Relative telomere length (rTL) was measured using quantitative polymerase chain reaction. The analysis showed associations between laboratory markers, oxidative stress markers and rTL in metabolically healthy and unhealthy participants. Total oxidation status (TOS), total antioxidant capacity (TAC) and rTL were significantly connected with metabolically unhealthy obesity. TAC was associated with metabolically healthy obesity. Telomeres shorten in patients with metabolic dysregulation related to oxidative stress and obesity linked to MetS. Further studies among young metabolically healthy and unhealthy individuals are needed to determine the pathways related to metabolic disturbances that cause oxidative stress that leads to MetS.

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