Effects of long-term ethanol storage of blood samples on the estimation of telomere length

Telomeres, DNA structures located at the end of eukaryotic chromosomes, shorten with each cellular cycle. The shortening rate is affected by factors associated with stress, and, thus telomere length has been used as a biomarker of ageing, disease, and different life history trade-offs. Telomere research has received much attention in the last decades, however there is still a wide variety of factors that may affect telomere measurements and to date no study has thoroughly evaluated the possible long-term effect of a storage medium on telomere measurements. In this study we evaluated the long-term effects of ethanol on relative telomere length (RTL) measured by qPCR, using blood samples of magpies collected over twelve years and stored in absolute ethanol at room temperature. We firstly tested whether storage time had an effect on RTL and secondly we modelled the effect of time of storage (from 1 to 12 years) in differences in RTL from DNA extracted twice in consecutive years from the same blood sample. We also tested whether individual amplification efficiencies were influenced by storage time, and whether this could affect our results. Our study provides evidence of an effect of storage time on telomere length measurements. Importantly, this effect shows a pattern of decreasing loss of telomere sequence with storage time that stops after approximate 4 years of storage, which suggests that telomeres may degrade in blood samples stored in ethanol. Our method to quantify the effect of storage time could be used to evaluate other storage buffers and methods. Our results highlight the need to evaluate the long-term effects of storage on telomere measurements, particularly in long-term studies.