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Abstract
Measurement of telomere length is widely used in epidemiologic studies. Insufficient standardization of the measurements processes has, however, complicated the comparison of results between studies. We aimed to investigate whether DNA extraction methods have an influence on measured values of relative telomere length (RTL) and whether this has consequences for epidemiological studies. We performed four experiments with RTL measurement in quadruplicate by qPCR using DNA extracted with different methods: 1) a standardized validation experiment including three extraction methods (magnetic-particle-method EZ1, salting-out-method INV, phenol-chloroform-isoamyl-alcohol PCI) each in the same 20 samples demonstrated pronounced differences in RTL with lowest values with EZ1 followed by INV and PCI-isolated DNA; 2) a comparison of 307 samples from an epidemiological study showing EZ1-measurements 40% lower than INV-measurements; 3) a matching-approach of two similar non-diseased control groups including 143 pairs of subjects revealed significantly shorter RTL in EZ1 than INV-extracted DNA (0.844 ± 0.157 vs. 1.357 ± 0.242); 4) an association analysis of RTL with prevalent cardiovascular disease detected a stronger association with INV than with EZ1-extracted DNA. In summary, DNA extraction methods have a pronounced influence on the measured RTL-values. This might result in spurious or lost associations in epidemiological studies under certain circumstances.
Highlights
Measurement of telomere length is widely used in epidemiologic studies
We recently observed in various studies we performed that differences in the range and level of relative telomere length (RTL) measurements might be influenced by factors other than phenotypical characteristics of the investigated patients or subjects[15]
This is in line with two small studies, which both recently proposed that the results of telomere length measurement by quantitative polymerase chain reaction (qPCR) and even Southern blotting might depend on the used DNA extraction method[27,28]
Summary
Measurement of telomere length is widely used in epidemiologic studies. Insufficient standardization of the measurements processes has, complicated the comparison of results between studies. Used techniques are the absolute measurement of telomere length with restriction fragments analysis by Southern blot[21,22] and the relative measurement by real-time quantitative polymerase chain reaction (qPCR)[23] The latter is a frequently used method in epidemiological studies since much less DNA is required and it is less laborious allowing a high-throughput approach. We recently observed in various studies we performed that differences in the range and level of RTL measurements might be influenced by factors other than phenotypical characteristics of the investigated patients or subjects[15] This is in line with two small studies, which both recently proposed that the results of telomere length measurement by qPCR and even Southern blotting might depend on the used DNA extraction method[27,28]. The central aim of the present in-depth investigation was to systematically compare the results of telomere length ascertainment by qPCR, the T/S-ratios, as a consequence of DNA extraction methods and assess its impact on epidemiological studies by four interconnected experiments
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