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.