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 qPCR repeatability (intra- and inter-assay levels) to be at similar levels across three common storage media (ethanol, Longmire's and Queen's). However, inter-extraction repeatability was 50% lower for samples stored in Queen's lysis buffer, indicating storage medium can influence precision. Precision as well as accuracy could be increased by estimating rTL from multiple qPCR reactions and from multiple extractions. Repetition increased statistical power equivalent to a 25% (single extraction analysed twice) and 17% (two extractions) increase in sample size. Overall, this study identifies novel sources of variability in high-throughput telomere quantification and provides guidance on sampling strategy design and how to increase rTL precision and accuracy.
The data package contains one dataset:
- This file contains several datasets from Eastwood et al. 2017 MER. Each tab represents a different analysis: Overall relative telomere length (rTL) repeatability, inter-assay rTL repeatability, inter-extraction rTL repeatability, intra-assay GAPDH Cq repeatability, intra-assay Telomere Cq repeatability, DNA extraction method rTL comparison, DNA extraction digestion temperature comparison (Cq values and relative telomere length) and a comparison between TRF and rTL methods. Details of data collection and column heading terms can be found in the manuscript.
- Molecular aging
- Statistical power