Blood and Dried Blood Spot Telomere Length Measurement by qPCR: Assay Considerations

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.

Telomere length change plateaus at 4 years of age in Latino children: associations with baseline length and maternal change

“…Telomere length measurements by qPCR were reported to be higher in dried blood spots than whole blood taken from both finger and arm venipuncture (Zanet et al 2013) suggesting systematic difference in qPCR-derived telomere length measurements using different specimen collection method. The reason for this systematic difference is currently unknown.…”

Early Telomere Shortening and Genomic Instability in Tubo-Ovarian Preneoplastic Lesions

“…BRCA2-mediated homologous recombination reactions contribute to the maintenance of telomere length by facilitating telomere replication (37,38). An association between shorter telomeres in peripheral blood leucocytes and increased risk of ovarian cancer, particularly for younger women, was reported in families carrying BRCA1/2 mutations as well as in sporadic cases (39). Our data suggest that BRCA1 mutations might have greater impact on telomere shortening than BRCA2 mutations in both ovarian and tubal dysplasia, and further studies are necessary to confirm this finding and to investigate possible underlying mechanisms.…”

Telomere length in children environmentally exposed to low-to-moderate levels of lead

“…The assessment of rTL and B-Pb were performed under strict quality control and followed MIQE guidelines (Bustin et al, 2009) and an inter-laboratory proficiency test, respectively. Additionally, the sampling and handling of blood were strictly controlled because blood proceeding appears to have a significant effect on telomere length (Zanet et al, 2013). However, although the mean European B-Pb in urban children is lower than the level of concern, which was established at 50 μg/l (CDC, 2012;Hruba et al, 2012), areas where this level doubles still exist (Hruba, unpublished) primarily near industrial emitters (lead smelters).…”