Abstract
PER2 gene methylation is closely related to the occurrence and progress of some cancers, but there is no method to quantitatively detect PER2 methylation in conventional laboratories. So, we established a TaqMan real-time fluorescence quantitative methylation specific PCR (TaqMan real-time FQ-MSP) assay and use it for quantitative detection of PER2 methylation in leukemia patients. According to the PER2 sequence searched by GenBank, a CpG sequence enrichment region of the PER2 gene promoter was selected, and the methylated and unmethylated target sequences were designed according to the law of bisulfite conversion of DNA to construct PER2 methylation positive and negative reference materials. Specific primers and probe were designed. The reference materials were continuously diluted into gradient samples by tenfold ratio to evaluate the analytical sensitivity, specificity, accuracy and reproducibility of the method, and the analytical sensitivity of TaqMan real-time FQ-MSP assay was compared with that of the conventional MSP assay. At the same time, the new-established TaqMan real-time FQ-MSP assay and the conventional MSP assay were used to detect the PER2 methylation level of 81 patients with leukemia, and the samples with inconsistent detection results of the two assays were sent to pyromethylation sequencing to evaluate the clinical detection performance. The minimum detection limit of TaqMan real-time FQ-MSP assay for detecting PER2 methylation level established in this study was 6 copies/uL, and the coefficient of variation(CV) of intra-assay and inter-assay was less than 3%. Compared with the conventional MSP assay, it has higher analytical sensitivity. For the samples with inconsistent detection results, the results of pyrosequencing and TaqMan real-time FQ-MSP assay are consistent. TaqMan real-time FQ-MSP assay of PER2 methylation established in this study has high detection performance and can be used for the detection of clinical samples.
Highlights
The Period 2 (PER2) gene is a member of the PER gene family composed of PER1, PER2 and PER3, and is one of the core genes that control the circadian rhythm[1]
The results showed that the minimum detection limit of conventional methylation-specific PCR (MSP) assay is 60 copies/uL, the minimum detection limit of qPCR is 60 copies/uL, and the minimum detection limit of TaqMan real-time TaqMan real-time fluorescence quantitative MSP (FQ-MSP) assay established in this study is 6 copies/uL (Fig. 4)
We have developed and verified a TaqMan real-time FQ-MSP assay that can detect PER2 methylation level, which effectively solves the problems of MSP and bisulfite sequencing (BSP)
Summary
The PER2 gene is a member of the PER gene family composed of PER1, PER2 and PER3, and is one of the core genes that control the circadian rhythm[1]. A large number of studies have shown that the abnormal expression of PER2 is closely related to the occurrence and development of a variety of tumors, such as breast cancer[3], ovarian cancer[4], pancreatic cancer[5], oral squamous cell carcinoma[6], liver cancer[7], large intestine cancer et al.[8]. The abnormal expression of PER2 can be detected in a variety of hematological malignancies[9]. Compared with mononuclear cells of normal human bone marrow and tonsil, the PER2 level of patients with acute myeloid leukemia (AML) is significantly downregulated[11]. PER2 gene methylation is closely related to the occurrence and progress of some cancers, but there is no method to quantitatively detect PER2 methylation in conventional laboratories. We established a TaqMan real-time fluorescence quantitative methylation specific PCR (TaqMan real-time FQ-MSP) assay and use it for quantitative detection of PER2 methylation in leukemia patients
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