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Real-time quantitative polymerase chain reaction assay for detecting the eps8 gene in acute myeloid leukemia
Lei Wang1, Song-Hao Cai1, Wen-Yan Xiong1
1Department of Hematology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
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.
