The telomere length measurement assay was adapted from the published original method by Cawthon (2 (link),3 (link)). The telomere thermal cycling profile consisted of: Cycling for T (telomeric) PCR: denature at 96°C for 1 s, anneal at 54°C for 60 s, with fluorescence data collection, 30 cycles. Cycling for S (single copy gene) PCR: denature at 95°C for 15 s, anneal at 58°C for 1 s, extend at 72°C for 20 s, eight cycles; followed by denature at 96°C for 1 s, anneal at 58°C for 1 s, extend at 72°C for 20 s, hold at 83°C for 5 s with data collection, 35 cycles.
The primers for the telomere PCR 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 primers for the single-copy gene (human beta-globin) PCR 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 contained 20 mM Tris–HCl, pH 8.4; 50 mM KCl; 200 µM each dNTP; 1% DMSO; 0.4× Syber Green I; 22 ng Escherichia coli DNA per reaction; 0.4 U of Platinum Taq DNA polymerase (Invitrogen Inc.) per 11 µl reaction; 0.5–10 ng of genomic DNA. Tubes containing 26, 8.75, 2.9, 0.97, 0.324 and 0.108 ng of a reference DNA (from Hela cancer cells) were included in each PCR run so that the quantity of targeted templates in each sample was determined relative to the reference DNA sample by the standard curve method. Each concentration of the reference DNA was run as quadruplets and samples were run as triplicates.
To control for inter-assay variability, eight control DNA samples from cancer cell lines were included in each run. The cell lines included 293T, H1299, UMUC3 and UMUC3 cells infected with a lentiviral construct containing the telomerase RNA gene to extend telomeres harvested at various population doublings after infection. In each assay batch, the T/S ratio of each control DNA was divided by the average T/S for the same DNA from 10 runs to obtain a normalizing factor. This was done for all eight samples and the average normalizing factor for all eight samples was used to correct the participant DNA samples to obtain the final T/S ratio.
The T/S ratio for each sample was measured twice. In this procedure, when the duplicate T/S value and the initial value varied by >7%, the sample was run a third time and the average of two closest values was reported. Typically, in cohorts from human clinical studies, ∼15% of samples have needed to be assayed the third time.
The primers for the telomere PCR 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 primers for the single-copy gene (human beta-globin) PCR 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 contained 20 mM Tris–HCl, pH 8.4; 50 mM KCl; 200 µM each dNTP; 1% DMSO; 0.4× Syber Green I; 22 ng Escherichia coli DNA per reaction; 0.4 U of Platinum Taq DNA polymerase (Invitrogen Inc.) per 11 µl reaction; 0.5–10 ng of genomic DNA. Tubes containing 26, 8.75, 2.9, 0.97, 0.324 and 0.108 ng of a reference DNA (from Hela cancer cells) were included in each PCR run so that the quantity of targeted templates in each sample was determined relative to the reference DNA sample by the standard curve method. Each concentration of the reference DNA was run as quadruplets and samples were run as triplicates.
To control for inter-assay variability, eight control DNA samples from cancer cell lines were included in each run. The cell lines included 293T, H1299, UMUC3 and UMUC3 cells infected with a lentiviral construct containing the telomerase RNA gene to extend telomeres harvested at various population doublings after infection. In each assay batch, the T/S ratio of each control DNA was divided by the average T/S for the same DNA from 10 runs to obtain a normalizing factor. This was done for all eight samples and the average normalizing factor for all eight samples was used to correct the participant DNA samples to obtain the final T/S ratio.
The T/S ratio for each sample was measured twice. In this procedure, when the duplicate T/S value and the initial value varied by >7%, the sample was run a third time and the average of two closest values was reported. Typically, in cohorts from human clinical studies, ∼15% of samples have needed to be assayed the third time.
