A quantitative PCR method for measuring absolute telomere length

Telomeres are nucleoprotein structures that cap the ends of chromosomes. The integrity of the telomere structure and its DNA hexamer (TTAGGG)n repeat sequence is critical for the protection of the ends of chromosomes from degradation and in maintaining overall genomic stability[1,2]. The number of DNA hexamer (TTAGGG)n repeats is reduced during each cell division in differentiated cells, and as a consequence telomere length (TL) often decreases in most differentiated cells throughout the lifespan of the organism [1]. Shortening of telomeres can result in telomere end fusions and an increased level of chromosome instability (CIN), which is in turn a key initiating event in numerous cancers (including lung, breast, colon, and prostate cancers, as well as certain leukaemia's)[3-7]. It has been shown that telomere shortening can be accelerated by environmental factors such as psychological and physiological stress, cigarette smoking, obesity and high homocysteine [8-14]. Efficiency of TL maintenance is also affected by gender[15-17]. TL has been shown to be associated prospectively with increased risk of myocardial infarction, coronary artery disease, breast cancer free survival, clear cell renal cell carcinoma survival, post-stroke mortality, dementia and cognitive decline, as well as total survival independent of genetic influences [18-24].For all of these reasons there has been a burgeoning interest in measuring TL accurately and efficiently to understand both the fundamental biology of telomere maintenance as well as determining the modifiable dietary and life-style factors that contribute substantially to accelerated TL attrition. A wide range of methods have been developed to measure TL such as (i) the gold standard Terminal Restriction Fragment (TRF) analysis by hybridisation of digested DNA with telomere sequence probes, (ii) Flow-FISH cytometry of cells following hybridisation with fluorescent peptide nucleic acid (PNA) probes, (iii) quantitative fluorescence i