SUPPLEMENTARY MATERIAL AND METHODS
Relative telomere length (RTL)
We used a quantitative PCR method [1, 2] to estimate the quantity of telomere repeated DNA sequences relative to a reference gene
(c-Myc) that was predetermined to be non-variable in copy number (non-VCN) among the sample genomes as per Smith et al. [3].
This ratio of telomere to reference (or normaliser) sequence was also standardised among runs by division by the same ratio for a
control sample (interplate calibrator) included in each PCR run (see equation below). Primer sequences for the non-VCN gene (5’GAG GGC CAA GTT GGA CAG TG-3’, and 5’-TTG CGG TTG TTG CTG ATC TG-3’) amplified a 54 bp portion of the c-Myc
proto-oncogene. Forward and reverse telomeric primers were 5’- CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG
GTT-3’ and 5’- GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT-3’, respectively [2].
Samples were diluted to a standard DNA concentration of ~20 ng μl-1. Reactions were prepared using the Qiagility PCR robot
(Qiagen, Germany) in 20 μl volumes and contained 10 μl SensiMix™ SYBR no-Rox Kit (Bioline, Germany), and 300 nM of each
primer for telomeres or 200 nM of each primer for non-VCN gene reactions. Cycling conditions included an initial hold at 95°C for 5
mins followed by 40 cycles of 94°C for 10 secs, 58°C for 15 secs, and 72°C for 20 secs with fluorescence readings recorded at this
step. A final melt step was included on each run with the temperature ramping from 65°C to 95°C in 1°C increments. Cycling was
performed on a Rotorgene 6000 real-time PCR instrument (Qiagen, Germany). In each of seven runs, we included three replicates of
DNA samples taken at each of the three time points from each individual, plus a no-template control (water) and five replicates of the
control DNA sample taken at the first sampling time from an individual that died during the first hibernation period and was not
included in the experiment. Multiple single-use aliquots of the extracted DNA from the control sample were stored at -20C until use
in each run (all seven runs were conducted over two days). As a measure of repeatability, the median coefficient of variation (CoV) of
the three replicate reactions for each sample after efficiency was raised to the power of the cycle threshold was 7.1%, with 72% of
samples having a CoV < 10%. We found that efficiency was systematically greater for reactions of the non-VCN gene sequence than
the telomere sequence (p = 0.02). We also detected a significant increase in the efficiency of reactions as a function of sample age (p <
0.01; Figure S1). Consequently, in calculations of RTL, we estimated individual reaction efficiency using the software LinRegPCR
(V.12.5) [4] and used an average value of efficiency for groups of same-aged samples within each amplicon.
Using the mean PCR efficiency per sample age and amplicon (E) and a cycle threshold (Cq) value per sample, we calculated
the RTL using the formula [5]:
RTLS = (ECT^CqCT / EST^CqST) / (ECN^CqCN / ESN^CqSN)
Where: RTLS = relative telomere length for a given sample; ECT = mean efficiency of the control telomere reactions; CqCT =
mean cycle threshold of the control telomere replicates; EST = mean efficiency of all sample telomere reactions; CqST = mean cycle
threshold of within-sample telomere replicates; ECN = mean efficiency of the control non-VCN gene reactions; CqCN = mean cycle
threshold of the control non-VCN gene replicates; ESN = average efficiency of all sample non-VCN gene reactions; and CqSN =
average cycle threshold of within-sample non-VCN gene replicates.
REFERENCES
1.
2.
3.
4.
5.
Cawthon, R.M. 2002 Telomere measurement by quantitative PCR. Nuc. Acids Res. 30, e47. (doi:10.1093/nar/30.10.e47).
Callicott, R.J. & Womack, J.E. 2006 Real-time PCR assay for measurement of mouse telomeres. Comp. Med. 56, 17-22.
Smith, S., Turbill, C. & Penn, D.J. 2011 Chasing telomeres, not red herrings, in evolutionary ecology. Heredity 107(4), 372373.
Ruijter, J.M., Ramakers, C., Hoogaars, W.M.H., Karlen, Y., Bakker O., van den Hoff, M.J.B. & Moorman, A.F.M. 2009
Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nuc. Acids Res.37, e45.
(doi:10.1093/nar/gkp045).
Ehrlenbach, S., Willeit, P., Kiechl, S., Willeit, J., Reindl, M., Schanda, K., Kronenberg, F. & Brandstaetter, A. 2009 Influences
on the reduction of relative telomere length over 10 years in the population-based Bruneck Study: introduction of a wellcontrolled high-throughput assay. Int. J. Epidem. 38, 1725-1734. (doi:10.1093/ije/dyp273).
telomere
non-VCN gene
Reaction efficiency
2.02
2.00
1.98
1.96
1.94
0
50
100
150
200
250
300
Age of extracted DNA sample (days)
Figure S1 Average reaction efficiency of each amplicon plotted as a function of the age of the sample of extracted DNA from ear
tissue of the edible dormouse, Glis glis.