Ravindranathan Ajay, Diolaiti Morgan E, Cimini Beth A, Stohr Bradley A
Department of Pathology, University of California, San Francisco, California.
UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California.
Curr Protoc Cell Biol. 2019 Dec;85(1):e97. doi: 10.1002/cpcb.97.
Telomerase plays a critical role in cancer and aging by adding hexa-nucleotide repeats to the ends of telomeres and extending the cellular proliferative lifespan. The very low level of telomerase expression in most cell populations and the difficulty of detecting telomere elongation in single cells have limited the study of telomerase expression and function in individual cells of a heterogeneous population. The method described in this article combines single-molecule detection (RNAscope) of telomerase reverse transcriptase (TERT) with our previously described TSQ1 assay for in situ monitoring of telomere extension, thereby enabling detection of TERT expression, telomere length, and telomere elongation in single cells and providing a unique approach for studying the factors that regulate telomere elongation by telomerase. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: TSQ1 lentivirus production Basic Protocol 2: TSQ1 lentiviral infection and plating Basic Protocol 3: RNAscope analysis Basic Protocol 4: TSQ1 PNA-FISH detection.
端粒酶通过在端粒末端添加六核苷酸重复序列并延长细胞增殖寿命，在癌症和衰老过程中发挥关键作用。大多数细胞群体中端粒酶表达水平极低，且难以在单细胞中检测到端粒延长，这限制了对异质群体中单个细胞中端粒酶表达和功能的研究。本文所述方法将端粒酶逆转录酶（TERT）的单分子检测（RNAscope）与我们先前描述的用于原位监测端粒延长的TSQ1检测相结合，从而能够在单细胞中检测TERT表达、端粒长度和端粒延长，并为研究调节端粒酶介导的端粒延长的因素提供了一种独特的方法。© 2019约翰威立国际出版公司。基本方案1：TSQ1慢病毒生产 基本方案2：TSQ1慢病毒感染及铺板 基本方案3：RNAscope分析 基本方案4：TSQ1肽核酸荧光原位杂交检测。
