Telomerase activity quantification qpcr assay kit

5.0 × 10⁶ cells were harvested and frozen at −80 °C. Thawed cells were treated with cell lysis buffer in Telomerase Activity Quantification qPCR Assay Kit (ScienCell, Carlsbad, CA, USA, 8928) supplemented with 100 μm phenylmethylsulfonyl fluoride in isopropanol and 0.03% (v/v) β‐mercaptoethanol and assayed following the instructions of the kit. qPCR was performed using OneStepPlus (Thermo Fisher Scientific).

Telomerase activity was determined by using Telomerase Activity Quantification qPCR Assay Kit (ScienCell Research Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. A total of 5 × 10⁴ cells per well with miRNA mimic or negative control were seeded into 24-well plates and cultured for 48 h. After lysing cells, the telomerase reactions were prepared for each sample and incubated at 37 °C for 3 h. The reaction was stopped by heating the samples to 85 °C for 10 min and the quantitative PCR (qPCR) was performed on the LightCycler^® 480 II Instrument (Roche, Basel, Switzerland). All the experiments were performed in triplicate.

Telomerase is a ribonucleic acid–protein complex composed of a single long non-coding RNA, called telomerase RNA, and associated proteins. NSCs telomerase activity was detected by Telomerase Activity Quantification qPCR Assay Kit (ScienCell, Carlsbad, CA, USA) that was amplified via quantitative real-time PCR (qPCR). Briefly, the cultured cells were lysed with lysis buffer supplemented with PMSF and β-mercaptoethanol. Then, the homogenized samples prepared were left at 4 °C for 30 min and were spun at 12,000–16,000 g for 20 min. Telomerase reactions (Cell lysate sample; 5× Telomerase reaction buffer; Nuclease-free H₂O) were incubated at 37 °C for 3 h and terminated by heating at 85 °C for 10 min. The qPCR reactions (Post telomerase reaction sample; Primer stock solution; qPCR master mix; Nuclease-free H₂O) were performed under the following conditions: 95 °C for 10 min, 95 °C for 20 s, 52 °C for 20 s, and amplified for 36 PCR cycles (72 °C for 45 s) with the LightCycler 480 II instrument (Roche Applied Science, Basel, Switzerland).

Telomerase activity was measured by telomerase activity quantification qPCR assay kit (ScienCell, Carlsbad, CA, USA #8928). Approximately 4 × 10⁶ cells for each replicate (N = 6) for both cell culture conditions were extracted and analyzed according to the product protocol. Briefly, cells were detached by trypsin, counted by trypan blue exclusion assay, and pelleted. Cell proteins were extracted by cell lysis buffer supplemented with PMSF 0.1 M in isopropanol and β-mercaptoethanol. After cell protein extraction telomerase reaction was performed as described in the product protocol. Finally, qPCR was done to analyze the telomer production by telomerase. ΔCq was calculated by the following equation: $\Delta C{q}_{ } = C{q}_{\left( CTRL \right)}- C{q}_{\left(Cholest-4,6-dien-3-one\right)}$
ΔCq is quantification cycle value obtained from qPCR.
The relative telomerase activity was calculated using the formula described above: $Relative telomerase activity = 2^{-\mathsf{\Delta }\mathrm{Cq}}$

Quantitative determination of telomerase activity was performed using Telomerase activity quantification qPCR assay kit (Sciencell research lab., Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol.