Liu Zhaojun, Zhou Jing, Gu Liankun, Deng Dajun
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Haidian District, Beijing, 100142, China.
Oncotarget. 2016 Aug 30;7(35):56447-56455. doi: 10.18632/oncotarget.10906.
Methylation changes of CpG islands can be determined using PCR-based assays. However, the exact impact of the amount of input templates (TAIT) on DNA methylation analysis has not been previously recognized. Using COL2A1 gene as an input reference, TAIT difference between human tissues with methylation-positive and -negative detection was calculated for two representative genes GFRA1 and P16. Results revealed that TAIT in GFRA1 methylation-positive frozen samples (n = 332) was significantly higher than the methylation-negative ones (n = 44) (P < 0.001). Similar difference was found in P16 methylation analysis. The TAIT-related effect was also observed in methylation-specific PCR (MSP) and denatured high performance liquid chromatography (DHPLC) analysis. Further study showed that the minimum TAIT for a successful MethyLight PCR reaction should be ≥ 9.4 ng (CtCOL2A1 ≤ 29.3), when the cutoff value of the methylated-GFRA1 proportion for methylation-positive detection was set at 1.6%. After TAIT of the methylation non-informative frozen samples (n = 94; CtCOL2A1 > 29.3) was increased above the minimum TAIT, the methylation-positive rate increased from 72.3% to 95.7% for GFRA1 and 26.6% to 54.3% for P16, respectively (Ps < 0.001). Similar results were observed in the FFPE samples. In conclusion, TAIT critically affects results of various PCR-based DNA methylation analyses. Characterization of the minimum TAIT for target CpG islands is essential to avoid false-negative results.
可以使用基于聚合酶链反应（PCR）的检测方法来确定CpG岛的甲基化变化。然而，输入模板量（TAIT）对DNA甲基化分析的确切影响此前尚未得到认识。以COL2A1基因为输入参考，计算了GFRA1和P16这两个代表性基因在甲基化检测呈阳性和阴性的人体组织之间的TAIT差异。结果显示，GFRA1甲基化阳性的冷冻样本（n = 332）中的TAIT显著高于甲基化阴性样本（n = 44）（P < 0.001）。在P16甲基化分析中也发现了类似的差异。在甲基化特异性PCR（MSP）和变性高效液相色谱（DHPLC）分析中也观察到了与TAIT相关的效应。进一步研究表明，当甲基化GFRA1比例的甲基化阳性检测临界值设定为1.6%时，成功进行甲基化荧光定量PCR（MethyLight PCR）反应的最低TAIT应≥9.4 ng（CtCOL2A1≤29.3）。将甲基化无信息的冷冻样本（n = 94；CtCOL2A1 > 29.3）的TAIT提高到最低TAIT以上后，GFRA1的甲基化阳性率分别从72.3%提高到95.7%，P16的甲基化阳性率从26.6%提高到54.3%（P < 0.001）。在福尔马林固定石蜡包埋（FFPE）样本中也观察到了类似结果。总之，TAIT严重影响各种基于PCR的DNA甲基化分析结果。确定目标CpG岛的最低TAIT对于避免假阴性结果至关重要。
