Real-Time Quantitative Polymerase Chain Reaction Assay for Detecting the Eps8 Gene in Acute Myeloid Leukemia
Real-Time Quantitative Polymerase Chain Reaction Assay for Detecting the Eps8 Gene in Acute Myeloid Leukemia
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DOI：
10.7754/clin.lab.2013.121107
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发表时间：
2013-01-01
影响因子：
0.7
通讯作者：
Li, Yu-Hua
中科院分区：
文献类型：
--
作者：
Wang, Lei;Cai, Song-Hao;Li, Yu-Hua
背景资料：Eps 8是一个新的原癌基因，与许多人类实体瘤的发生、增殖、转移、化疗耐药和预后有关。然而，Eps 8在急性髓系白血病（AML）中的功能仍不清楚。方法：扩增Eps 8基因，将其克隆到pMD 18-T中，构建重组质粒pMD 18-T/Eps 8，作为建立实时定量PCR方法的标准DNA。结果：该标准品的最低检测限为100拷贝，灵敏度是常规PCR的10倍，具有较高的灵敏度。熔解曲线分析表明，所设计的引物具有特异性和可操作性。与对照组相比，AML患者Eps 8的表达水平升高（p = 0.013）。结论：本实验建立的检测方法可用于AML患者Eps 8表达水平的检测。结果表明，Eps 8可以作为急性髓细胞白血病患者化疗反应性的预后因素。
Background: Eps8 is a novel proto-oncogene related to the tumorigenesis, proliferation, metastasis, chemo-resistance, and prognosis of many human solid cancers. However, the function of Eps8 in acute myeloid leukemia (AML) is still unclear. Thus, this study aims to develop a real-time quantitative polymerase chain reaction (PCR) assay for Eps8 in AML.Methods: Eps8 was amplified and cloned into pMD18-T to generate the recombinant plasmid pMD18-T/Eps8 as standard DNA for the establishment of real-time quantitative PCR. This assay was used to detect the expression level of Eps8 in bone marrow samples from AML patients and healthy volunteers (control group).Results: The limit of detection achieved using this standard was 100 copies, which was 10 times more sensitive than that achieved using conventional PCR, indicating high sensitivity. Melting curve analysis demonstrated that the primers designed for the established real-time quantitative PCR assay were specific and available. The expression level of Eps8 in the AML patients increased compared with that in the control group (p = 0.013). Furthermore, the expression level of Eps8 showed significant correlation with the complete remission rate of AML patients to chemotherapy (p = 0.024).Conclusions: The established assay is useful in detecting the expression level of Eps8. The results suggest that Eps8 may serve as a prognostic factor of responsiveness to chemotherapy in AML patients.