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.
Highlights
Telomeres are nucleoprotein structures that cap the ends of chromosomes
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]
The protocol we describe is a modification to Cawthon’s quantitative realtime polymerase chain reaction (qPCR) based relative quantification (Telomere/Single Copy Gene ratio) method by introducing an oligomer standard to generate absolute telomere length (aTL) values
Summary
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]. 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 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. The protocol we describe is a modification to Cawthon’s qPCR based relative quantification (Telomere/Single Copy Gene ratio) method by introducing an oligomer standard to generate aTL values. CRITICAL: SCG amplification is crucial for the accuracy of the results generated in the aTL qPCR assay; changes in amount of template that is present in each reaction can by affected by pipetting or DNA quantification error. Optical PCR plates and caps Centrifuge for spinning plates Real-Time PCR machine (e.g. Applied Biosystems 7300) Software for analyzing data (e.g. Applied Biosystems SDS software v1.4) Software for analyzing data (e.g. Microsoft Excel, SPSS)
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