2. Telomere measurement methods
DNA was extracted from whole blood samples using Macherey Nagel
Nucleospin Blood kits following the manufacturer’s protocol. Avian red
blood cells are nucleated and are well suited for longitudinal telomere
analysis (Nussey et al. 2014). DNA concentration and purity were
assessed with a Nanodrop 8000 (Thermo Scientific). Relative telomere
length was measured using qPCR (quantitative polymerase chain reaction)
on an Mx3000P (Stratagene) as described in Cawthon (2002) and modified
for use in common gulls (Rattiste et al., 2015). The relative telomere
length (T/S) of the samples was calculated as the ratio of the telomere
repeat copy number (T) to that of a single copy control gene (S),
relative to the reference sample. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as the single copy control gene. We used
the following gull specific GAPDH forward and reverse primers
(Integrated DNA Technologies): 5’-CGGAGCACCGCTTACAATTT-3’ and 5’-
GCATCTCCCCACTTGATGTTG-3’ respectively. Amplified samples were run on a
3% agarose gel to verify that the amplification was a single product,
which yielded a single band at 77 bp as expected. We used the following
telomere primers (Quanta Bio, with final concentration of 200 nM): TEL
1b: 5’-CGGTTTGTTTGGGTTTGGGTTTGGGTT-3’ and TEL 2b:
5’-GGCTTGCCTTACCCTTACCCTTACCCT-3. The qPCR reactions for GAPDH and
telomeres were run on separate plates. All reactions used 20 ng of DNA
in a final volume of 25 µl containing 12.5 µl of SYBER green Master Mix,
0.25 µl forward and reverse primer, 6 µl water, and 6 µl of DNA sample.
A negative control of water was run on each plate. All samples were run
in duplicate, and average values were used to determine the T/S ratio.
Treatment groups were distributed approximately equally between plates.
Replicates of each sample, and the first and second sample for each bird
were run on the same plate. In order to assess the efficiencies of each
plate, samples were run against a standard curve of 40, 20, 10, 5, and
2.5 ng produced by serially diluting a reference sample. In all cases,
plate efficiencies were in the accepted range (i.e. 100+/-15%) and all
of the samples fell within the bounds of the standard curve. Average
plate efficiencies and standard errors for GAPDH and telomere plates
were 102.34 ± 3.00, and 98.94 ± 3.00, respectively. The average
intra-plate variation of the Ct values was 0.82% for the telomere
assays and 0.19% for the GAPDH assays, and the inter-plate CV-s of the
Ct values for telomere and GAPDH assays were 0.64% and 0.34%,
respectively. The same individual was also included on every plate and
the coefficient of variation of the T/S ratio across plates was 8.12%.
A subset of samples (45) was ran twice for assessing measurement
accuracy. Average Intraclass Correlation Coefficient (ICC) in this
subset was 0.934 (95% CI 0.879 ± 0.964), p < 0.0001, for
single measurements ICC = 0.876 (95% CI 0.785 ± 0.930) p <
0.0001.