Different qPCR master mixes influence telomere primer binding within and between bird species.

The analysis of telomere dynamics in birds is a growing research field providing important findings on ecological and environmental variations in individuals' aging, fitness and lifespan. Real‐time quantitative PCR (qPCR) has gained much interest for the evaluation of telomere length in birds. However, the assessment of several key preanalytical and analytical factors to optimize the method for achieving reproducible results, and the influence of these factors on the conclusions of each study, have been generally overlooked. In this study, we assessed the performance of eight commercially‐available qPCR master mixes in the amplification of telomere fragments in two bird species (zebra finch Taeniopygia guttata and red‐billed chough Pyrrhocorax pyrrhocorax). We observed that qPCR master mixes influence the telomere primer binding to target sequences and the amplification specificity within and between bird species, although PCR amplification efficiencies were very close to 100% for all master mixes. These findings indicated that the suitability of the master mix and other analytical factors must be carefully evaluated before starting a telomere dynamics study, especially when the technique has not previously been used in the species. We also showed that optimal PCR amplification efficiencies do not translate to good qPCR telomere assays, and that inference about amplification specificity based only on melting curve data can lead to misleading conclusions. Overall, this work highlights the complexity of qPCR optimization for the study of telomere length in birds. [ABSTRACT FROM AUTHOR]