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)).