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Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood

Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly...

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Autores principales: Tolios, Alexander, Teupser, Daniel, Holdt, Lesca M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670203/
https://www.ncbi.nlm.nih.gov/pubmed/26636575
http://dx.doi.org/10.1371/journal.pone.0143889
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author Tolios, Alexander
Teupser, Daniel
Holdt, Lesca M.
author_facet Tolios, Alexander
Teupser, Daniel
Holdt, Lesca M.
author_sort Tolios, Alexander
collection PubMed
description Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.
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spelling pubmed-46702032015-12-10 Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood Tolios, Alexander Teupser, Daniel Holdt, Lesca M. PLoS One Research Article Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements. Public Library of Science 2015-12-04 /pmc/articles/PMC4670203/ /pubmed/26636575 http://dx.doi.org/10.1371/journal.pone.0143889 Text en © 2015 Tolios 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Tolios, Alexander
Teupser, Daniel
Holdt, Lesca M.
Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood
title Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood
title_full Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood
title_fullStr Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood
title_full_unstemmed Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood
title_short Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood
title_sort preanalytical conditions and dna isolation methods affect telomere length quantification in whole blood
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670203/
https://www.ncbi.nlm.nih.gov/pubmed/26636575
http://dx.doi.org/10.1371/journal.pone.0143889
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