Bleeding protocol and telomere measurements
We collected blood (10-30µl) at age 5, 12 and 80 days, from the jugular or brachial vein of 75 nestlings (36 controls and 39 heated) using a hypodermic needle and capillary tube. Blood was taken from all individuals of each clutch and preserved in 1ml of 95% ethanol, stored at room temperature in an Eppendorf tube, and labeled with a unique alphanumeric code.
We extracted genomic DNA (gDNA) from blood samples using the NucleoSpin blood kit (Macherey-Nagel) with some minor modifications. 2 µl of whole blood were removed from the sample tube, allowed to air –dry evaporating the ethanol, and subsequently added to 198µl of PBS. From this stage, we followed the manufacturer’s protocol, with the gDNA eluted into 35µl of BE buffer and stored at -20oC awaiting telomere length analysis. The quantity and purity of the gDNA was measured on a Nanodrop 8000 and all samples were within the accepted parameters; A260/280 ≥1.8, A260/230 ≥ 1.9 (Thermo Fisher).
We assayed rTL using the qPCR method (Criscuolo 2009). Briefly, the rTL 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 pooled DNA reference sample from five zebra finches at age 12 days that was run on all plates. We used Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 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 the telomere and the control gene (mean ± SE, TEL: 87.14 ± 1.30%; GAPDH: 106.39 ± 1.12%). Average inter-plate variation of the Ct values was 1.58 for the telomere assay and 0.42 for the GAPDH assay. Intra-plate coefficient of variation for the telomere and GAPDH assays for the raw Ct values were 13.59 and 24.31 respectively. The average intra-plate variation of the Ct values was 0.8 for the telomere assay and 0.41 for the GAPDH assay.
rTL measurements were calculated using the method by Pfaffl (2001). The mean values were used to calculate the rTL (T:S ratio) using the formula: ((1 + E telomere)^ ΔCq telomere (control – sample) / (1 + E GAPDH)^ ΔCq GAPDH (control – sample)).