Telomerase Activity Quantification in Ovarian Samples

Ovarian samples were homogenized using RNAse-free pestle and mortar and lysed as in [74 (link)]. The CY5-labelled telomerase-substrate primer (TS-primer: 5′-Cy5-AATCCGTCGAGCAGAGTT-3′, Sigma-Aldrich [75 (link)]); was elongated, and elongation products were amplified together with an internal control, as in [75 (link)]. Two protein concentrations were used for each sample (0.5 and 2.5 μg). A negative control was included by preincubating each sample extract with RNase (Roche Diagnosis) for 10 min at 30°C as in [76 (link)]. Jurkat cells were used as a positive control. Electrophoresis was run in an acrylamide: bisacrylamide 19:1 gel (Bio-Rad) using Protean II (Bio-Rad) electrophoresis chambers. Gels with Cy5 signals were imaged wet in ChemiDoc (Bio-Rad). Image Lab software (version 5.0) was used for quantification of the TRAP image shown in Figure 2.