Abstract
Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for differences in RTL measurements from samples extracted by different DNA extraction methods within a study.
Highlights
Telomere shortening has recently been identified as one of nine ‘hallmarks of aging’ [1] and blood cell telomere length (TL) is an increasingly widely measured biomarker in human epidemiology and vertebrate ecology [2,3,4]
We addressed the effect of DNA extraction method on relative TL (RTL) measurements by comparing two silica membrane-based kits (SC and SP) with a kit that uses a non-membrane-based salting out method (PG)
This study adds to the emerging literature showing that DNA extraction methods may affect the mean RTL measurement produced by Quantitative PCR (qPCR) techniques
Summary
Telomere shortening has recently been identified as one of nine ‘hallmarks of aging’ [1] and blood cell telomere length (TL) is an increasingly widely measured biomarker in human epidemiology and vertebrate ecology [2,3,4]. There is mounting recent evidence that relative TL (RTL) measurements by qPCR may be influenced by methods of sample acquisition and storage [9] and DNA extraction methods [10,11,12,13,14]. Understanding how such methodological variation may influence RTL measurements by qPCR both within and among laboratories is essential for evaluating and comparing results of telomere studies
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