Telomere Length Quantification in Cancer Cells

DNA was extracted from cancer cells using Wizard Genomic DNA Purification Kit (Promega, WI, USA) according to manufacturer’s protocol, and stored at −20 °C. Telomere length was assessed using two pairs of primers i.e. telomere-specific and a single copy gene-specific (albumin), as previously described^{43 (link)–45 (link)}. Briefly, we used the primers that were already shown to work specifically with conditions providing efficiency close to 100%. Initial denaturation and polymerase activation (hot start) was performed in 95 °C for 5 min. The signal was detected during 45 cycles i.e. 95 °C/10 s, 60 °C/15 s and 72 °C/10 s. Melting analysis (65–95 °C range, 0.11 °C resolution) at the end of the reaction melting analysis was performed to verify specificity of the product. The telomere length was assessed using a LightCycler 96 qPCR system (Roche, Germany) and FastStart Sybr Green Master (Roche, Germany).