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      A quantitative PCR method for measuring absolute telomere length

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      1 , , 1
      Biological Procedures Online
      BioMed Central

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          Abstract

          We describe a simple and reproducible method to measure absolute telomere length (aTL) using quantitative real-time polymerase chain reaction (qPCR). This method is based on the Cawthon method for relative measurement of telomere length (TL) but modified by introducing an oligomer standard to measure aTL. The method describes the oligomer standards, the generation of the standard curve and the calculations required to calculate aTL from the qPCR data. The necessary controls and performance characteristics of the assay are described in detail and compared relative to other methods for measuring TL. Typical results for this assay for a variety of human tissue samples are provided as well as a troubleshooting schedule. This method allows high throughput measurement of aTL using small amounts of DNA making it amenable for molecular epidemiological studies. Compared to the traditional relative TL qPCR assays, the aTL method described in this protocol enables a more direct comparison of results between experiments within and between laboratories.

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          Most cited references23

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          Oxidative stress shortens telomeres.

          Telomeres in most human cells shorten with each round of DNA replication, because they lack the enzyme telomerase. This is not, however, the only determinant of the rate of loss of telomeric DNA. Oxidative damage is repaired less well in telomeric DNA than elsewhere in the chromosome, and oxidative stress accelerates telomere loss, whereas antioxidants decelerate it. I suggest here that oxidative stress is an important modulator of telomere loss and that telomere-driven replicative senescence is primarily a stress response. This might have evolved to block the growth of cells that have been exposed to a high risk of mutation.
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            Obesity, cigarette smoking, and telomere length in women.

            Obesity and smoking are important risk factors for many age-related diseases. Both are states of heightened oxidative stress, which increases the rate of telomere erosion per replication, and inflammation, which enhances white blood cell turnover. Together, these processes might accelerate telomere erosion with age. We therefore tested the hypothesis that increased body mass and smoking are associated with shortened telomere length in white blood cells. We investigated 1122 white women aged 18-76 years and found that telomere length decreased steadily with age at a mean rate of 27 bp per year. Telomeres of obese women were 240 bp shorter than those of lean women (p=0.026). A dose-dependent relation with smoking was recorded (p=0.017), and each pack-year smoked was equivalent to an additional 5 bp of telomere length lost (18%) compared with the rate in the overall cohort. Our results emphasise the pro-ageing effects of obesity and cigarette smoking.
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              A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes.

              A highly conserved repetitive DNA sequence, (TTAGGG)n, has been isolated from a human recombinant repetitive DNA library. Quantitative hybridization to chromosomes sorted by flow cytometry indicates that comparable amounts of this sequence are present on each human chromosome. Both fluorescent in situ hybridization and BAL-31 nuclease digestion experiments reveal major clusters of this sequence at the telomeres of all human chromosomes. The evolutionary conservation of this DNA sequence, its terminal chromosomal location in a variety of higher eukaryotes (regardless of chromosome number or chromosome length), and its similarity to functional telomeres isolated from lower eukaryotes suggest that this sequence is a functional human telomere.
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                Author and article information

                Journal
                Biol Proced Online
                Biological Procedures Online
                BioMed Central
                1480-9222
                2011
                31 January 2011
                : 13
                : 3
                Affiliations
                [1 ]CSIRO Food and Nutritional Sciences, Nutritional Genomics Laboratory, PO Box 10041, Adelaide BC, SA, 5000, Australia
                Article
                1480-9222-13-3
                10.1186/1480-9222-13-3
                3047434
                21369534
                c31d76fd-877b-4b00-ac52-f7228b5d9aa2
                Copyright ©2011 O'Callaghan and Fenech; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 7 October 2010
                : 31 January 2011
                Categories
                Methodology

                Life sciences
                Life sciences

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