Increasing the accuracy and precision of relative telomere length estimates by RT qPCR

Since attrition of telomeres, DNA caps that protect chromosome integrity, is accelerated by various forms of stress, telomere length (TL) has been proposed as an indicator of lifetime accumulated stress. In ecological studies it has been used to provide insights into aging, life-history trade-offs, the costs of reproduction and disease. qPCR is a high throughput and cost effective tool to measure relative TL (rTL) that can be applied to newly-collected and archived ecological samples. However, qPCR is susceptible to error both from the method itself and pre-analytical steps. Here, repeatability was assessed overall and separately across multiple levels (intra-assay, inter-assay and inter-extraction) to elucidate the causes of measurement error, as a step towards improving precision. We also tested how accuracy, defined as the correlation between the ‘gold standard’ for TL estimation (telomere restriction fragment length analysis with in-gel hybridisation), could be improved. We find qPC...