Telomere measurements
Genomic DNA (gDNA) was extracted using the NucleoSpin blood kit (Macherey-Nagel) with some minor modifications. 2 µl of whole blood was removed from the sample tube and allowed to air-dry, to evaporate the ethanol, and subsequently added to 198µl of PBS. From this stage, the manufacturer’s protocol was followed, with the gDNA eluted into 35µl of BE buffer. The quantity and purity of the gDNA was measured on a Nanodrop 8000 and all samples were within the accepted parameters; A260/280 ≥1.7, A260/230 ≥ 1.8 (Thermo Fisher). The gDNA was stored at -20oC until telomere length analysis was performed.
Telomere length was assayed using the qPCR method as described previously (Criscuolo 2009). Briefly, the telomere length of each sample was measured by determining the ratio (T:S) of telomere repeat copy number (T) to a single copy or non-variant control gene (S), relative to a five pooled DNA reference sample from wild zebra finches at day 11 that was run on all plates. For this study, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the control gene. A standard curve (6 serial dilutions of zebra finch gDNA from 40 to 1.25ng/well) was also included on each plate and all samples (ran in triplicate) fell within standard curve boundaries. Mean reaction efficiencies were within the acceptable range for telomere and control gene (mean ± SE, TEL: 97.43 ± 4.54%; GAPDH: 87.73 ± 3.75%). The average inter‐plate variation of the Ct values was 1.47 for the telomere assay and 2.6 for the GAPDH assay. Intra-plate coefficient of variation for the telomere and GAPDH assays for the raw Ct values were 12.44 and 27.08 respectively . The average intra-plate variation of the Ct values was 0.74 for the telomere assay and 0.47 for the GAPDH assay.
Telomere length measurements were calculated using the method by Pfaffl (2001). The mean values were used to calculate the telomere length (T:S ratio) using the formula: ((1 + E telomere)^ ΔCq telomere (control – sample) / (1 + E GAPDH)^ ΔCq GAPDH (control – sample)).
In addition we had sufficient DNA from a small number of 11 day samples (5) that enabled us to measure absolute telomere length using the in-gel TRF method (see Nussey et al., 2014 for details of this method). This enabled us to compare telomere length in wild and captive zebra finches.