Measurement of telomere length is crucial for the study of telomere maintenance and its role in molecular pathophysiology of diseases and in aging. Several methods are used to measure telomere length, the choice of which usually depends on the type and size of sample to be assayed, as well as cost and throughput considerations. The goal of this study was to investigate the factors that may influence the reliability of qPCR-based relative telomere length measurements in whole blood. Day to day intra-individual variability, types of blood anticoagulant, sample storage conditions, processing and site of blood draw were investigated. Two qPCR-based methods to measure telomere length (monoplex vs. multiplex) were also investigated and showed a strong correlation between them. Freezing and thawing of the blood and storage of the blood at 4°C for up to 4 days did not affect telomere length values. Telomere lengths in dried blood spots were significantly higher than both whole blood and peripheral mononuclear blood cells, and were highly correlated with both. We found that telomere length measurements were significantly higher in dried blood spots collected directly from fingertip prick compared to dried blood spots prepared with anticoagulated whole blood collected from the finger, and non-blotted whole blood taken from both finger and arm venipuncture. This suggests that DNA from cells blotted on paper is not equivalent to that collected from venipuncture whole blood, and caution should be taken when comparing between blood sample types.

Telomere length (TL) measurement is central to many biomedical research, population, and epidemiology studies, with promising potential as a clinical tool. Various assays are used to determine TL, depending on the type and size of the sample. We describe the detailed optimization of a monochromatic multiplex realtime quantitative PCR (MMqPCR) assay for relative TL using the LightCycler 480. MMqPCR was initially developed using a different instrument with many separate reagents. Differences in instrument performance, reagents, and workflow required substantial optimization for the assay to be compatible with the LightCycler 480. We optimized the chemistry of the assay using a purchased one-component reaction mix and herein describe sources of variability and quality control relevant to the MMqPCR TL assay on any instrument. Finally, the assay was validated against other TL assays, such as terminal restriction fragment, Southern blot, and flow fluorescent in situ hybridization. The correlations obtained between data from MMqPCR and these assays (R 2 Z 0.88 and 0.81) were comparable to those seen with the monoplex version (R 2 Z 0.85 and 0.82) when the same samples were assayed. The intrarun and interrun CV ranged from 4.2% to 6.2% and 3.2% to 4.9%, respectively. We describe a protocol for measuring TL on the LightCycler platform that provides a robust high-throughput method applicable to clinical diagnostics or large-scale studies of archived specimens.

Cellular response to thymidine analogue NRTI exposure showed a complex, time- and dose-dependent pattern over time. We report for the first time that NRTIs can induce MDR-1 expression; however, this effect is delayed, possibly in response to oxidative damage or mitochondrial dysfunction. Our results indicate that longitudinal experiments may refine our knowledge about NRTI toxicity.

There is limited evidence regarding the potential risk of untargeted iron supplementation, especially among individuals who are iron-replete or have genetic hemoglobinopathies. Excess iron exposure can increase the production of reactive oxygen species, which can lead to cellular damage. We evaluated the effect of daily oral supplementation on relative leukocyte telomere length (rLTL) and blood mitochondrial DNA (mtDNA) content in non-pregnant Cambodian women (18–45 years) who received 60 mg of elemental iron as ferrous sulfate (n = 190) or a placebo (n = 186) for 12 weeks. Buffy coat rLTL and mtDNA content were quantified by monochrome multiplex quantitative polymerase chain reaction. Generalized linear mixed-effects models were used to predict the absolute and percent change in rLTL and mtDNA content after 12 weeks. Iron supplementation was not associated with an absolute or percent change in rLTL after 12 weeks compared with placebo (ß-coefficient: −0.04 [95% CI: −0.16, 0.08]; p = 0.50 and ß-coefficient: −0.96 [95% CI: −2.69, 0.77]; p = 0.28, respectively). However, iron supplementation was associated with a smaller absolute and percent increase in mtDNA content after 12 weeks compared with placebo (ß-coefficient: −11 [95% CI: −20, −2]; p = 0.02 and ß-coefficient: −11 [95% CI: −20, −1]; p= 0.02, respectively). Thus, daily oral iron supplementation for 12 weeks was associated with altered mitochondrial homeostasis in our study sample. More research is needed to understand the risk of iron exposure and the biological consequences of altered mitochondrial homeostasis in order to inform the safety of the current global supplementation policy.