荧光实时定量PCR检测HULClncRNA方法的建立-第三军医大学学报.doc

实时荧光定量PCR检测HULC lncRNA方法的建立及初步应用 汪先桃 郭变琴 梁勤东 涂植光 (重庆医科大学检验医学院 临床检验诊断学教育部重点实验室 重庆 400016) ［摘　要］目的：建立实时荧光定量PCR检测长链非编码RNA HULC的方法，探讨其在作为肝癌标志的应用价值。方法：根据Genebank中的HULC lncRNA(AY914050.1)全序列, 设计HULC基因的特异引物, 构建HULC基因的T克隆载体，命名为pMD18-T-HULC；以pMD18-T-HULC为实时荧光定量PCR检测HULC的标准品，建立实时荧光定量PCR检测HULC的方法并进行方法学评价。应用此方法检测8种肿瘤细胞株、肝癌组织、其它癌及癌旁组织中HULC lncRNA的表达。结果：HULC实时荧光定量PCR检测方法学的最低检测限为1.8×103拷贝/L, 线性范围为1.8×103拷贝/L~4.6×109拷贝/L；该法的批内变异系数（CV）2.0%, 批间CV8.0%。其在肝癌组织和肝癌细胞株起始表达量为(7.6±3.1)×105拷贝/L ~(5.6±3.2)×106拷贝/L，明显高于其它癌和癌旁组织HULC lncRNA表达量（1.8×103拷贝/L）。新发现膀胱癌细胞株T24中表达量高[(6.7±2.4)×105拷贝/L]，但在膀胱癌组织中表达低（1.8×103拷贝/L）。结论：成功建立了实时荧光定量PCR检测HULC lncRNA的方法，HULC lncRNA可望作为肝癌的标志。 ［关键词］HULC；lncRNA；实时荧光定量PCR；肝癌 [中图法分类号]　R446.9; R34;R73-31　　　　［文献标志码］Ａ Establishment of the method of real-time fluorescence quantitative PCR detection of HULC lncRNA and its primary application WANG Xiantao, GUO Bianqin, LIANG Qindong, TU Zhiguang* (College of Laboratory Medicine, Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing 400016, China) [Abstract] Objective To establish a real-time fluorescent quantitative PCR for method the detection of long non coding RNA HULC (highly up-regulated in liver cancer), and to explore its application value of hepatocellular carcinoma marker. Methods Specific primers for HULC lncRNA gene were designed according to full HULC lncRNA sequence in Genbank (AY914050.1). The T cloning vector for the quantitative standard of HULC gene detection was constructed and named pMD18-T-HULC. The real-time fluorescence quantitative PCR method of HULC was established and methodologically evaluated. The primary application of this method to detect HULC lncRNA in 8 types of tumor cell lines, and liver cancer and other cancer tissues was investigated. Results The minimum detection limit of established HULC real-time fluorescent quantitative PCR detection method was 1.8×103 copies /L, the linear range was 1.8×103 copies /L ~4.6 ×109 copies /L, the in