Leukocyte telomere length analysis
Extracted DNA was shipped in batch to the Blackburn Telomere Lab at the University of California, San Francisco. The telomere qPCR primers were tel1b [5′-CGGTTT(GTTTGG)5GTT-3′], used at a final concentration of 100 nM, and tel2b [5′-GGCTTG(CCTTAC)5CCT-3′], used at a final concentration of 900 nM. The single-copy gene (human beta-globin) qPCR primers were hbg1 [5′-GCTTCTGACACAACTGTGTTCACTAGC-3′], used at a final concentration of 300 nM, and hbg2 [5′-CACCAACTTCATCCACGTTCACC-3′], used at a final concentration of 700 nM. The final reaction mix consisted of the following: 20 mM Tris-hydrochloride, pH 8.4; 50 mM potassium chloride; 200 μM each deoxyribonucleotide triphosphate; 1% dimethyl sulfoxide; 0.4 × SYBR green I; 22 ng The thermal profile for telomere (T) consisted of denaturing at 96 °C for 1 min followed by 30 cycles of denaturing at 96 °C for 1 s and annealing or extension at 54 °C for 60 s with fluorescence data collection. The thermal profile for a single copy gene (S) consisted of denaturing at 96 °C for 1 min followed by 8 cycles of denaturing at 95 °C for 15 s, annealing at 58 °C for 1 s, and extension at 72 °C for 20 s, followed by 35 cycles of denaturing at 96 °C for 1 s, annealing at 58 °C for 1 s, extension at 72 °C for 20 s, and holding at 83 °C for 5 s with data collection. The T/S ratio for each sample was measured in duplicate runs, each with triplicate wells. When the duplicate T/S values disagreed by more than 7%, the sample was run in triplicate and the two closest values were used. The Dixon Q test was used to remove outliers from the triplicates. The average of the T or S concentrations from the remaining wells was used to calculate the T/S ratio for each sample. This is repeated to get the run 2 T/S ratios. If the difference between run 1 and run 2 larger than 7%, a third run was performed and the average of the 2 closest values was used as the final data. Potential batch difference was adjusted by running combined plates with a small set of samples from each plate. All samples used the same lot of reagents for LTL assay. The average inter-assay coefficient of variation (CV) for this study was 2.2%. The PCR efficiencies were 96.2 ± 1.6% and 98.0 ± 2.9% for the telomere reactions (T) and S reactions (S) respectively. All samples were processed in the Blackburn Telomere Lab at UCSF. We did not calculate the interclass correlation coefficient (ICC) on repeat DNA extractions for this study because they were assayed before this recommendation was made by the Telomere Research Network.