Guo Guang-Hong, Xie Yi-Bin, Jiang Tao, An Yang
Department of Laboratory Medicine, The First Medical Center of Chinese PLA General Hospital, Beijing 100853, China.
Department of Pancreatic and Gastric Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
World J Gastrointest Oncol. 2022 Oct 15;14(10):2038-2047. doi: 10.4251/wjgo.v14.i10.2038.
Gastric cancer (GC) is one of the most prevalent malignant tumors that endangers human health. Early diagnosis is essential for improving the prognosis and survival rate of GC patients. Ring finger protein 180 (RNF180) is involved in the regulation of cell differentiation, proliferation, apoptosis, and tumorigenesis, and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC. Thus, methylated RNF180 can be used as a potential biomarker for GC diagnosis.
To use droplet digital polymerase chain reaction (ddPCR) to quantify the methylation level of the RN180 gene. A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.
The primer and probe were designed and selected, the conversion time of bisulfite was optimized, the ddPCR system was adjusted by primer concentration, amplification temperature and amplification cycles, and the detection limit of ddPCR was determined.
The best conversion time for blood DNA was 2 h 10 min, and that for plasma DNA was 2 h 10 min and 2 h 30 min. The results of ddPCR were better when the amplification temperature was 56 °C and the number of amplification cycles was 50. Primer concentrations showed little effect on the assay outcome. Therefore, the primer concentration could be adjusted according to the reaction system and DNA input. The assay required at least 0.1 ng of input DNA.
In summary, a ddPCR assay was established to detect methylated RNF180, which is expected to be a new diagnostic biomarker for GC.
胃癌（GC）是危害人类健康的最常见恶性肿瘤之一。早期诊断对于改善GC患者的预后和生存率至关重要。环状泛素连接酶180（RNF180）参与细胞分化、增殖、凋亡和肿瘤发生的调控，启动子区域CpG岛的异常高甲基化与GC的发生发展密切相关。因此，甲基化的RNF180可作为GC诊断的潜在生物标志物。
使用液滴数字聚合酶链反应（ddPCR）定量RNF180基因的甲基化水平。设计并优化了一种可重复的ddPCR检测方法，用于从微量DNA中检测甲基化的RNF180。
设计并筛选引物和探针，优化亚硫酸氢盐转化时间，通过引物浓度、扩增温度和扩增循环数调整ddPCR体系，并确定ddPCR的检测限。
血液DNA的最佳转化时间为2小时10分钟，血浆DNA的最佳转化时间为2小时10分钟和2小时30分钟。扩增温度为56℃、扩增循环数为50时，ddPCR结果更佳。引物浓度对检测结果影响较小。因此，可根据反应体系和DNA输入量调整引物浓度。该检测方法至少需要0.1 ng输入DNA。
总之，建立了一种检测甲基化RNF180的ddPCR检测方法，有望成为GC的新型诊断生物标志物。
