Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR
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...
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| Format: | Online |
| Language: | English |
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Public Library of Science
2016
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056729/ |
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pubmed-5056729 |
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pubmed-50567292016-10-27 Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR Seeker, Luise A. Holland, Rebecca Underwood, Sarah Fairlie, Jennifer Psifidi, Androniki Ilska, Joanna J. Bagnall, Ainsley Whitelaw, Bruce Coffey, Mike Banos, Georgios Nussey, Daniel H. Research Article 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. Public Library of Science 2016-10-10 /pmc/articles/PMC5056729/ /pubmed/27723841 http://dx.doi.org/10.1371/journal.pone.0164046 Text en © 2016 Seeker et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Seeker, Luise A. Holland, Rebecca Underwood, Sarah Fairlie, Jennifer Psifidi, Androniki Ilska, Joanna J. Bagnall, Ainsley Whitelaw, Bruce Coffey, Mike Banos, Georgios Nussey, Daniel H. |
| spellingShingle |
Seeker, Luise A. Holland, Rebecca Underwood, Sarah Fairlie, Jennifer Psifidi, Androniki Ilska, Joanna J. Bagnall, Ainsley Whitelaw, Bruce Coffey, Mike Banos, Georgios Nussey, Daniel H. Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR |
| author_facet |
Seeker, Luise A. Holland, Rebecca Underwood, Sarah Fairlie, Jennifer Psifidi, Androniki Ilska, Joanna J. Bagnall, Ainsley Whitelaw, Bruce Coffey, Mike Banos, Georgios Nussey, Daniel H. |
| author_sort |
Seeker, Luise A. |
| title |
Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR |
| title_short |
Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR |
| title_full |
Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR |
| title_fullStr |
Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR |
| title_full_unstemmed |
Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR |
| title_sort |
method specific calibration corrects for dna extraction method effects on relative telomere length measurements by quantitative pcr |
| description |
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. |
| publisher |
Public Library of Science |
| publishDate |
2016 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056729/ |
| _version_ |
1613678166367272960 |