Zhang Hua-hua, Zeng Yao-yin, He Xian-hui, Xing Fei-yue
Institute of Tissue Transplantation and Immunology, Ji'nan University, Guangzhou 510632, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Jan;26(1):62-5.
To establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of naive T cells and evaluating the thymic function.
The genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG(2)) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C(57)BL/6 mice) were detected for sjTRECs by real-time quantitative PCR.
The standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains.
Real-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.
建立一种实时定量PCR方法，用于检测小鼠胸腺细胞和脾淋巴细胞中信号连接T细胞受体切除环（sjTRECs）的水平，以确定初始T细胞的数量并评估胸腺功能。
从小鼠胸腺细胞和脾细胞中提取基因组DNA，用于靶片段的PCR扩增。PCR产物纯化后，将重组激活基因2（RAG(2)）片段克隆到pGEMT-Easy载体中构建标准质粒。经过PCR优化后，获得标准曲线，并通过实时定量PCR检测样本（BALB/c和C(57)BL/6小鼠的胸腺细胞和脾细胞）中的sjTRECs。
成功构建了标准质粒，并获得了可靠性高的标准曲线。两种小鼠品系的T淋巴细胞中sjTREC含量无统计学差异。
成功建立了用于sjTREC分析的实时定量PCR方法，为胸腺功能分析提供了重要手段，为胸腺研究建立了可靠的模型。
