Telomere extraction and assay
We measured relative telomere length (RTL) from blood samples of
sparrows after they fledged, collected from 2000 to 2015. DNA sample
concentration was first measured using a Nanodrop 8000 Spectrophotometer
(Thermo Fisher) and normalized to 20–30 ng/µl. Next we used a
monochrome multiplex quantitative polymerase chain reaction (MMqPCR)
method to quantify RTL (Cawthon, 2009) as described in Chik et al.
(2023). In brief, MMqPCR quantifies RTL as a ratio of telomeric signals
to that of a single-copy reference gene (GAPDH in our study), where
amplification of both target sequences occur within a single well to
eliminate error from sample loading. Samples were allocated to plates
using a slicing approach (van Lieshout et al., 2020), where each ‘slice’
contained samples obtained from the same year, and each plate contained
samples from three consecutive slices, to avoid confounding plate and
sample year effects. We measured samples in duplicates in adjacent
wells. Plates were run using two machines, a QuantStudio 12K Flex
Real-Time PCR System (Thermo Fisher Scientific, five plates) and a
StepOnePlus (Applied Biosystems, 77 plates), and by two technicians (MEM
ran 52 plates and NdR ran 30 plates), but machine identity did not have
an effect on RTL (Sibma, 2021). After MMqPCR, we removed samples with Ct
values > 25, and a between-duplicate relative difference
> 0.2. We averaged the RTL of the remaining duplicates as
the final measure for each sample. Mean qPCR amplification efficiencies
for telomeres and the reference gene were 90.7% (range: 71 – 109%)
and 94.5% (range 76 – 119%) respectively (Sibma, 2021). The
inter-plate repeatability was 0.49 (s.e. = 0.07), while the intra-plate
repeatability was 0.98 (Sibma, 2021). The final telomere dataset
consisted of 2,083 telomere length measurements from 1,225 birds, 476 of
which have telomere length measurements at multiple ages.