Establishment of RT-qPCR method for telomerase catalytic subunits of mesenchymal stem cells*

CHEN Xiao-fei, LI Hui-ting, DONG Ying-ying, CAO Yi-dan, FU Xin-yue, LIU Ming-yue, ZHANG Rui-rui, WANG Yan-hui, WANG Xin-yue, CUI Meng-shan, ZHANG Tong, PANG Lin, RAO Chun-ming

Chinese Journal of Pharmaceutical Analysis ›› 2025, Vol. 45 ›› Issue (1) : 20-29.

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Chinese Journal of Pharmaceutical Analysis ›› 2025, Vol. 45 ›› Issue (1) : 20-29. DOI: 10.16155/j.0254-1793.2024-1333
Special Column for Quality Research and Evaluation of Stem Cell Products

Establishment of RT-qPCR method for telomerase catalytic subunits of mesenchymal stem cells*

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Abstract

Objective: To establish a simple and efficient method for detecting telomerase activity for evaluating tumorigenic risk of cell products. Methods: Specific primers and probes were designed for the conserved domain of telomerase catalytic subunit (TERT), and the primers and probes of TERT gene were optimized and screened, and the primers and probes of internal reference genes were set up. Multiple quantitative PCR reaction was performed using two-color fluorescent probes in a reaction system, and RT-qPCR probe method was established. The expression of TERT gene in human mesenchymal stem cells HMSC was used to determine whether telomerase activity existed in the cells indirectly. Results: TERT gene of 293T/17 positive control cells could be stably and specifically detected by this method, with a mean Ct value of 23.96 and a RSD of 1.5. The internal reference gene GAPDH could be detected successfully, the mean Ct value was 14.13, the RSD was 1.3%. The reference gene GAPDH in MRC-5 could be detected in negative control cells, the mean Ct value was 12.81, the RSD was 0.46%, the TERT gene was not detected, and the telomerase activity was negative. The reference gene GAPDH in HMSC could be detected, the Ct mean value was 13.01, and the RSD was 3.8%, whereas, the telomerase activity of HMSC was negative. Conclusion: The real-time fluorescent quantitative RT-qPCR probe method established in this study can accurately detect the expression of catalytic subunit mRNA in telomerase positive cells with good repeatability and high specificity. It can be used to analyze telomerase activity of stem cells and indirectly evaluate tumorigenic risk of cell products derived from stem cells.

Key words

fluorescent quantitative RT-PCR probe method / telomerase reverse transcriptase / telomerase activity / stem cell / tumorigenicity

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CHEN Xiao-fei, LI Hui-ting, DONG Ying-ying, et al. Establishment of RT-qPCR method for telomerase catalytic subunits of mesenchymal stem cells*[J]. Chinese Journal of Pharmaceutical Analysis, 2025, 45(1): 20-29 https://doi.org/10.16155/j.0254-1793.2024-1333

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