Method specific calibration corrects for DNA extraction method effects on relative telomere length measurements by quantitative PCR

LA Seeker, R Holland, S Underwood, J Fairlie, A Psifidi, JJ Ilska, A Bagnall, B Whitelaw, MP Coffey, G Banos, DH Nussey

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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.
Original languageEnglish
JournalPLoS ONE
Volume11 (10)
Issue numbere0164046
Early online date10 Oct 2016
DOIs
Publication statusFirst published - 10 Oct 2016

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telomeres
Telomere
Calibration
quantitative polymerase chain reaction
calibration
Polymerase Chain Reaction
DNA
methodology
Membranes
Silicon Dioxide
sampling
silica
Biomarkers
Soay
salting
Blood

Bibliographical note

1025115

Cite this

Seeker, LA., Holland, R., Underwood, S., Fairlie, J., Psifidi, A., Ilska, JJ., ... Nussey, DH. (2016). Method specific calibration corrects for DNA extraction method effects on relative telomere length measurements by quantitative PCR. PLoS ONE, 11 (10)(e0164046). https://doi.org/10.1371/journal.pone.0164046
Seeker, LA ; Holland, R ; Underwood, S ; Fairlie, J ; Psifidi, A ; Ilska, JJ ; Bagnall, A ; Whitelaw, B ; Coffey, MP ; Banos, G ; Nussey, DH. / Method specific calibration corrects for DNA extraction method effects on relative telomere length measurements by quantitative PCR. In: PLoS ONE. 2016 ; Vol. 11 (10), No. e0164046.
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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.",
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Seeker, LA, Holland, R, Underwood, S, Fairlie, J, Psifidi, A, Ilska, JJ, Bagnall, A, Whitelaw, B, Coffey, MP, Banos, G & Nussey, DH 2016, 'Method specific calibration corrects for DNA extraction method effects on relative telomere length measurements by quantitative PCR', PLoS ONE, vol. 11 (10), no. e0164046. https://doi.org/10.1371/journal.pone.0164046

Method specific calibration corrects for DNA extraction method effects on relative telomere length measurements by quantitative PCR. / Seeker, LA; Holland, R; Underwood, S; Fairlie, J; Psifidi, A; Ilska, JJ; Bagnall, A; Whitelaw, B; Coffey, MP; Banos, G; Nussey, DH.

In: PLoS ONE, Vol. 11 (10), No. e0164046, 10.10.2016.

Research output: Contribution to journalArticle

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AU - Seeker, LA

AU - Holland, R

AU - Underwood, S

AU - Fairlie, J

AU - Psifidi, A

AU - Ilska, JJ

AU - Bagnall, A

AU - Whitelaw, B

AU - Coffey, MP

AU - Banos, G

AU - Nussey, DH

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