2.8. qPCR absolute telomere length measurement

Telomere length was calculated by means of Absolute Human Telomere Length Quantification qPCR Assay Kit (ScienCell). The kit provided a primer solution for telomere amplification and another one that recognizes and amplifies a 100 base pair region on human chromosome 17. This last primer solution was used as single copy reference (SCR). Twenty nanograms of target DNA was added to a reaction containing the pair of primers (telomere or SCR) and FastStart Essential DNA Green Master (Roche), in a total reaction volume of 20µl, according to the manufacturer's instructions. PCR experiments were carried out on a Stratagene Mx3005P system (Agilent Technologies) and analyzed with MxPro 4.01 QPCR software Stratagene (Agilent Technologies). Telomere and SCR reactions were run in the same 96‐well plate and followed the same qPCR program setup (initial denaturation step at 95ºC for 10 minutes, followed by 32 cycles of 95°C for 20 seconds, 52ºC for 20 seconds and 72°C for 42 seconds, with signal acquisition).

Data were collected from duplicate reactions for each sample (cell lines, patients, and healthy donors). Duplicate values were accepted when the standard deviation of Ct was below 0.5 among replicates. The provided reference genomic DNA sample with known telomere length in kilobases served as reference to calculate samples’ telomere length (2^−∆∆Ct). The final result represents the average telomere length per chromosome.