Department of Oncology, The GIBH Affiliated Fuda Hospital, Chinese Academy of Sciences, Guangzhou, People's Republic of China.
Biotechnol Appl Biochem. 2012 Jan-Feb;59(1):55-63. doi: 10.1002/bab.69. Epub 2012 Feb 5.
Cell adhesion molecule CD44v6 has been found to be associated with the progression and metastasis of numerous cancers. In this study, a novel duplex real-time quantitative reverse-transcription PCR (qRT-PCR) assay was developed to quantitatively detect the CD44v6 gene expression in pancreatic cancer patients. The primers and probes of CD44v6 and β-actin genes were designed and standard curve of the duplex qRT-PCR was constructed by optimizing the reaction conditions. The specificity and reproducibility of this assay were satisfactory and the detection limit was 100 copies, which was 10 times more sensitive than the conventional RT-PCR assay. This assay was also used to detect the expression levels of CD44v6 messenger RNA in peripheral blood mononuclear cell in 37 pancreatic cancer patients and 12 healthy people. The results showed that 37 clinical samples were tested positive by the duplex qRT-PCR compared with only 30 by the conventional RT-PCR. The levels of CD44v6 expression showed significant correlation with sex, tumor size, tumor differentiation, clinical stage, lymph node, and liver metastasis (P < 0.05). Compared with the control group, CD44v6 levels in patients prior and 10 days post cryosurgery were significantly increased (P < 0.05) but had no significant change in those 1 month post cryosurgery (P > 0.05). The duplex qRT-PCR assay may provide a useful tool for the evaluation of prognosis and curative effect of pancreatic cancer.
细胞黏附分子 CD44v6 与许多癌症的进展和转移有关。在这项研究中，开发了一种新的双路实时定量逆转录 PCR（qRT-PCR）检测方法，用于定量检测胰腺癌患者中 CD44v6 基因的表达。设计了 CD44v6 和 β-肌动蛋白基因的引物和探针，并通过优化反应条件构建了双路 qRT-PCR 的标准曲线。该检测方法具有良好的特异性和可重复性，检测限为 100 拷贝，比常规 RT-PCR 检测方法灵敏 10 倍。该检测方法还用于检测 37 例胰腺癌患者和 12 例健康人外周血单个核细胞中 CD44v6 信使 RNA 的表达水平。结果显示，与常规 RT-PCR 相比，双路 qRT-PCR 检测到 37 例临床样本阳性。CD44v6 表达水平与性别、肿瘤大小、肿瘤分化、临床分期、淋巴结和肝转移显著相关（P<0.05）。与对照组相比，冷冻手术后 10 天患者的 CD44v6 水平明显升高（P<0.05），但冷冻手术后 1 个月无明显变化（P>0.05）。双路 qRT-PCR 检测方法可能为评估胰腺癌的预后和疗效提供有用的工具。
