Detection of Telomerase activity

Tissue extraction and PCR-ELISA were performed according to the instructions for the Telomerase PCR-ELISA kit (Roche Diagnostics, Mannheim, Germany). Briefly, 12 frozen sections (10 ym) of each sample was homogenized in 200 yL of ice cold lysis buffer and incubated for 30 minutes on ice. After centrifugation at 16,000g for 20 minutes at 4C, the supernatant was collected, quickly frozen in liquid nitrogen, and stored at -80 C. Protein concentration was measured using the DC protein assay kit (Bio-Rad Laboratories). Tissue extracts were incubated with a reaction buffer including a biotin labeled P1-TS primer and P2 primer, telomerase substrate, and Taq polymerase for 30 minutes at 25 °C in a final volume of 50 yL. After further incubation at 94 C for 5 minutes, the resulting mixture was subjected to the PCR for 30 cycles of 30 seconds at 94 C, 30 seconds at 50 C, and 90 seconds at 72 C.

The amplification products were denatured and hybridized with a digoxigenin labeled, telomeric repeat specific detection probe. The resulting product was immobilized through the biotin labeled TS primer to a streptavidin coated microtitre plate and detected with an antidigoxi- genin antibody conjugated with peroxidase. Absor- bance values were measured using a microtitre reader at 450 nm with a reference wavelength of 690 nm. Samples were regarded as telomerase posi­tive if the difference in absorbance was higher than 0.2 arbitrary units (A450-A690 nm). The absor- bance reading obtained with the positive control supplied with the kit was always higher than 1.5 U. All extracts were initially tested at 1 yg/yL of protein. In cases where telomerase activity was undetectable at this concentration, extracts were also tested at various concentrations ranging from 0.02 to 6 yg/yL of protein. The results were ex­pressed with relative telomerase activity (RTA). All assays performed in triplicate.  canadian pharmacy cialis

Statistical analysis

The statistical significance of differences in P.D and RTA were tested using the Student’s t-test. p-values<0.05 were considered statistically signifi­cant.