Absolute standards as a useful addition to the avian quantitative PCR telomere assay

Bird populations provide excellent systems to investigate variation in longevity in the wild since individuals can often be monitored over their lifetime. A number of recent studies suggest that the dynamics of protective telomere chromosome caps (telomere length and rate of loss) are indicative of biological state and potentially useful as indicators of future longevity. Currently, Terminal Restriction Fragment (TRF) analysis and relative quantitative PCR (qPCR) are used to measure telomeres in birds, but with limitations. TRF analysis is time consuming, while relative qPCR gives a within-study relative value making it difficult to compare across experiments. Utilising an approach first developed in humans of using synthetic oligomer telomeric (TTAGGG)n and normaliser gene standards of known length to calibrate qPCR values, we describe a methodological adaptation to the avian qPCR telomere assay to make results comparable within, and potentially between, bird species. We evaluate this absolute qPCR method in the Seychelles warbler Acrocephalus sechellensis against relative qPCR measurements on the same samples. Telomere estimates from both methods showed an age-related decline in telomere length, and were highly correlated (r = 0.99). Absolute qPCR avian telomere analysis may prove a useful means of estimating telomere lengths in a calibrated, sensitive, and efficient way using small amounts of archived bird sample.