Telotaggg telomerase pcr elisa kit

To analyze the telomerase activity of hUCB-MSCs quantitatively, we conducted a telomeric repeat amplification protocol assay using a TeloTAGGG Telomerase PCR ELISA kit (Roche Molecular Biochemicals, Brussels, Belgium) according to the manufacturer's protocol. The telomerase activity of hUCB-MSCs at passage 3 was measured and compared between two groups. Briefly, 2 × 10⁵ hUCB-MSCs were pelleted at 3000 g for 10 minutes at 4°C, washed twice with cold PBS, incubated for 20 minutes at 4°C with 200 μL of precooled lysis buffer (solution 1 of the kit), and centrifuged at 16,000 g for 20 minutes. Telomeric repeats were added to a biotin-labeled primer during the first reaction, and then, the elongation products were amplified by PCR. Finally, the immobilized PCR product was detected with an anti-digoxigenin-peroxidase antibody and visualized as a colored reaction product with the substrate 3,3′,5,5′-tetramethyl benzidine. The absorbance was measured in triplicate at 450 nm, by reading against a blank (reference absorbance at 690 nm). Samples were regarded as telomerase-positive if the difference in absorbance (A₄₅₀ − A₆₉₀) was greater than 0.2 [49 (link), 50 (link)].

Average telomere length was analyzed by Southern blotting analysis and at the level of the single cell by FISH. The genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and the Wizard^® Genomic DNA Purification Kit (Promega, Madison, WI, USA) for patients’ samples and murine cells, respectively, according to the manufacturer’s protocols. Terminal restriction fragments (TRF) length was measured by Southern blot method, using TeloTAGGG telomere length assay kit (Roche, Basel, Switzerland) following the manufacturer's protocol as described previously (Wnuk et al. 2014 (link)). In single cells, metaphase chromosomes were prepared as described elsewhere (Deregowska et al. 2020 (link)), and then Q-FISH was performed with the Telomere PNA FISH kit/Cy3 (Dako, Glostrup, Denmark) according to the protocol provided by the manufacturer’s recommendation.
Telomerase activity (TA) was measured with a TeloTAGGG Telomerase PCR ELISA kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

Cancer cells were processed according to the manufacturer's protocol for the TeloTAGGG Telomerase PCR ELISA kit (Roche, Orange, CA. Catalog number: 11854666910). Briefly, cell pellets were thawed in lysis reagent, incubated on ice for 30 minutes, and centrifuged at 16,000 g for 20 minutes at 4°C. Telomerase activity was immediately measured in the resultant supernatant using the telomeric repeat amplification protocol in which telomerase, if present in the cell lysate, adds telomeric repeats to the 3′ end of a biotin-labeled synthetic P1-TS primer. Samples were amplified by polymerase chain reaction (PCR), with P1-TS and P2 primers creating an elongated telomere. The PCR product was denatured and hybridized to a digoxigenin-labeled probe that detects telomeric repeats in a subsequent enzyme-linked immunosorbent assay (ELISA). Telomerase assays were performed three times independently and P values less than 0.05 were considered statistically significant.