title: Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR.
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.
authors.affiliation: Animal & Veterinary Sciences, SRUC, Roslin Institute Building, Easter Bush, Midlothian EH25 9RG, United Kingdom.|The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
authors.forename: Luise A
authors.identifier: http://orcid.org/0000-0001-6375-6372
authors.initials: LA
authors.lastname: Seeker
authors.affiliation: Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Midlothian, United Kingdom.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Holland
authors.affiliation: Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Midlothian, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Underwood
authors.affiliation: Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Midlothian, United Kingdom.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Fairlie
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
authors.forename: Androniki
authors.initials: A
authors.lastname: Psifidi
authors.affiliation: Animal & Veterinary Sciences, SRUC, Roslin Institute Building, Easter Bush, Midlothian EH25 9RG, United Kingdom.
authors.forename: Joanna J
authors.initials: JJ
authors.lastname: Ilska
authors.affiliation: SRUC Crichton Royal Farm, Glencaple Road, Dumfries, United Kingdom.
authors.forename: Ainsley
authors.initials: A
authors.lastname: Bagnall
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
authors.forename: Bruce
authors.initials: B
authors.lastname: Whitelaw
authors.affiliation: Animal & Veterinary Sciences, SRUC, Roslin Institute Building, Easter Bush, Midlothian EH25 9RG, United Kingdom.
authors.forename: Mike
authors.initials: M
authors.lastname: Coffey
authors.affiliation: Animal & Veterinary Sciences, SRUC, Roslin Institute Building, Easter Bush, Midlothian EH25 9RG, United Kingdom.|The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
authors.forename: Georgios
authors.initials: G
authors.lastname: Banos
authors.affiliation: Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Midlothian, United Kingdom.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Nussey
mesh_terms: D000818:Animals; D002138:Calibration; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012756:Sheep; D016615:Telomere / Q000235:genetics*; D059505:Telomere Homeostasis
chemical_list: D004247:DNA