Faculty Opinions recommendation of Influence of DNA extraction methods on relative telomere length measurements and its impact on epidemiological studies.
Year: 2016
Type: peer-review
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.Telomeres and telomerase discovered several decades ago are considered a "protection machinery" of our genome [1][2][3][4] .Telomeres have been under intensive investigation due to the hypothesis that they may be responsible for aging on the cellular level and affect lifespan 5,6 .Many independent cross-sectional studies postulate an association of short telomere length (TL) with higher risk for various diseases such as cancer and atherosclerosis including its comorbidities.Additionally, shorter TL has been related to mortality 7 and a variety of diseases [8][9][10][11][12][13][14][15][16][17] .Recently, data from prospective cohort studies including TL measurement at two different time points became available.From these studies evidence is accumulating that TL dynamics are not a one-way road with shortening over time [18][19][20] .Lengthening of telomeres can occur as well, which was observed in a large proportion (44% of 4,576 individuals) of the general population 20 .Altogether, a sinusoidal behavior of telomere length over time can be observed which decreases on average with age.Telomere length can be measured by different methods.Widely 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.Therefore, for all of our studies, we applied the well-established and as automated as possible high-throughput qPCR method for measurement of relative telomere length (RTL) with a high level of standardization to ensure reliable and high quality data for epidemiological studies.We generally perform RTL measurements in quadruplicate to maximize accuracy.
Author: Jerry Shay
Institution: The University of Texas Southwestern Medical Center
Language: English
Subfield: Physiology
Field: Medicine
Domain: Health Sciences
Open Access status: green
Awards: Verbundprojekt: Beobachtungsstudie zur chronischen Niereninsuffizienz in Deutschland (GCKD) Studie - TP2 Regionalzentren (anteilig), Verbundprojekt: Beobachtungsstudie zur chronischen Niereninsuffizienz in Deutschland (GCKD-Studie) - TP1 Studienzentrale, TP2 Regionalzentren (anteilig) und TP4 Substudie Koronarkalzifizierung (anteilig), Verbundprojekt: Beobachtungsstudie zur chronischen Niereninsuffizienz in Deutschland (GCKD-Studie) - TP2 Regionalzentren (anteilig) und TP4 Substudie Koronarkalzifizierung (anteilig), Verbundprojekt: Beobachtungsstudie zur chronischen Niereninsuffizienz in Deutschland (GCKD Studie) - TP2 Regionalzentren (anteilig)
OpenAlex ID: W4247195892