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Abstract
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.
Highlights
Telomeres are DNA-protein structures found at the end of linear eukaryotic chromosomes [1]
the average of duplicate S (T/S) should theoretically be independent of DNA concentration, in practice, it can diverge from linearity at extreme concentrations
To determine the assay’s variability, the intra-assay (n = 10) and inter-assay (n = 38) coefficient of variation (CV) for S, T and T/S values were calculated for three internal controls (IC) (Table 1)
Summary
Telomeres are DNA-protein structures found at the end of linear eukaryotic chromosomes [1]. Telomeric DNA consists of a highly conserved repetitive DNA sequence (TTAGGG)n, approximately 10–15 Kb in length, that does not code for proteins, but is associated with telomere-binding proteins and is of critical importance to cells [2,3]. Telomere functions include regulation of cellular replicative capacity, as well as stabilization and protection of chromosomal ends [4]. Telomere length (TL) shortening has been associated with aging, stress, increased risk for many age-associated conditions and diseases [5,6]. Leukocyte telomere length (LTL) has been reported to predict mortality and cardiovascular events [7]. There is great interest in measuring TL in various tissues, most commonly in blood
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