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Leukocyte telomere length variation due to DNA extraction method.

Denham J, Marques FZ, Charchar FJ - BMC Res Notes (2014)

Bottom Line: Shorter telomeres have been associated with several disease and health states.Telomeres extracted using the Lahiri and Nurnberger method (mean T/S ratio: 2.43, range: 1.57-3.02) and PureLink Genomic DNA Mini Kit (mean T/S ratio: 2.57, range: 2.24-2.80) did not differ (P=0.13).DNA purity was associated with telomere length.

View Article: PubMed Central - PubMed

Affiliation: Federation University Australia, Ballarat, Victoria, Australia. f.charchar@federation.edu.au.

ABSTRACT

Background: Telomere length is indicative of biological age. Shorter telomeres have been associated with several disease and health states. There are inconsistencies throughout the literature amongst relative telomere length measured by quantitative PCR (qPCR) and different extraction methods or kits used. We quantified whole-blood leukocyte telomere length using the telomere to single copy gene (T/S) ratio by qPCR in 20 young (18-25 yrs) men after extracting DNA using three common extraction methods: Lahiri and Nurnberger (high salt) method, PureLink Genomic DNA Mini kit (Life Technologies) and QiaAmp DNA Mini kit (Qiagen). Telomere length differences of DNA extracted from the three extraction methods was assessed by one-way analysis of variance (ANOVA).

Results: DNA purity differed between extraction methods used (P=0.01). Telomere length was impacted by the DNA extraction method used (P=0.01). Telomeres extracted using the Lahiri and Nurnberger method (mean T/S ratio: 2.43, range: 1.57-3.02) and PureLink Genomic DNA Mini Kit (mean T/S ratio: 2.57, range: 2.24-2.80) did not differ (P=0.13). Likewise, QiaAmp and Purelink-extracted telomeres were not statistically different (P=0.14). The Lahiri-extracted telomeres, however, were significantly shorter than those extracted using the QiaAmp DNA Mini Kit (mean T/S ratio: 2.71, range: 2.32-3.02; P=0.003). DNA purity was associated with telomere length.

Conclusion: There are discrepancies between the length of leukocyte telomeres extracted from the same individuals according to the DNA extraction method used. DNA purity could be responsible for the discrepancy in telomere length but this will require validation studies. We recommend using the same DNA extraction kit when quantifying leukocyte telomere length by qPCR or when comparing different cohorts to avoid erroneous associations between telomere length and traits of interest.

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Linear correlation between telomere assays. Telomere assays were repeated on 80% of samples on a separate day to assess the reproducibility of the data. Telomere length (T/S ratio) data showed acceptable reproducibility (r = 0.82, P < 0.001).
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Fig1: Linear correlation between telomere assays. Telomere assays were repeated on 80% of samples on a separate day to assess the reproducibility of the data. Telomere length (T/S ratio) data showed acceptable reproducibility (r = 0.82, P < 0.001).

Mentions: The inter-assay coefficient of variation between independent replicates for the T/S ratio was 9.7% and r-value was 0.82 (P < 0.001, Figure 1). The intra-assay coefficient of variation was 1.5% for the telomere primer-set and 0.75% for the 36B4 primer-set, respectively. Therefore, the data showed acceptable reproducibility.Figure 1


Leukocyte telomere length variation due to DNA extraction method.

Denham J, Marques FZ, Charchar FJ - BMC Res Notes (2014)

Linear correlation between telomere assays. Telomere assays were repeated on 80% of samples on a separate day to assess the reproducibility of the data. Telomere length (T/S ratio) data showed acceptable reproducibility (r = 0.82, P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289347&req=5

Fig1: Linear correlation between telomere assays. Telomere assays were repeated on 80% of samples on a separate day to assess the reproducibility of the data. Telomere length (T/S ratio) data showed acceptable reproducibility (r = 0.82, P < 0.001).
Mentions: The inter-assay coefficient of variation between independent replicates for the T/S ratio was 9.7% and r-value was 0.82 (P < 0.001, Figure 1). The intra-assay coefficient of variation was 1.5% for the telomere primer-set and 0.75% for the 36B4 primer-set, respectively. Therefore, the data showed acceptable reproducibility.Figure 1

Bottom Line: Shorter telomeres have been associated with several disease and health states.Telomeres extracted using the Lahiri and Nurnberger method (mean T/S ratio: 2.43, range: 1.57-3.02) and PureLink Genomic DNA Mini Kit (mean T/S ratio: 2.57, range: 2.24-2.80) did not differ (P=0.13).DNA purity was associated with telomere length.

View Article: PubMed Central - PubMed

Affiliation: Federation University Australia, Ballarat, Victoria, Australia. f.charchar@federation.edu.au.

ABSTRACT

Background: Telomere length is indicative of biological age. Shorter telomeres have been associated with several disease and health states. There are inconsistencies throughout the literature amongst relative telomere length measured by quantitative PCR (qPCR) and different extraction methods or kits used. We quantified whole-blood leukocyte telomere length using the telomere to single copy gene (T/S) ratio by qPCR in 20 young (18-25 yrs) men after extracting DNA using three common extraction methods: Lahiri and Nurnberger (high salt) method, PureLink Genomic DNA Mini kit (Life Technologies) and QiaAmp DNA Mini kit (Qiagen). Telomere length differences of DNA extracted from the three extraction methods was assessed by one-way analysis of variance (ANOVA).

Results: DNA purity differed between extraction methods used (P=0.01). Telomere length was impacted by the DNA extraction method used (P=0.01). Telomeres extracted using the Lahiri and Nurnberger method (mean T/S ratio: 2.43, range: 1.57-3.02) and PureLink Genomic DNA Mini Kit (mean T/S ratio: 2.57, range: 2.24-2.80) did not differ (P=0.13). Likewise, QiaAmp and Purelink-extracted telomeres were not statistically different (P=0.14). The Lahiri-extracted telomeres, however, were significantly shorter than those extracted using the QiaAmp DNA Mini Kit (mean T/S ratio: 2.71, range: 2.32-3.02; P=0.003). DNA purity was associated with telomere length.

Conclusion: There are discrepancies between the length of leukocyte telomeres extracted from the same individuals according to the DNA extraction method used. DNA purity could be responsible for the discrepancy in telomere length but this will require validation studies. We recommend using the same DNA extraction kit when quantifying leukocyte telomere length by qPCR or when comparing different cohorts to avoid erroneous associations between telomere length and traits of interest.

Show MeSH