Significant impact of amount of PCR input templates on various PCR-based DNA methylation analysis and countermeasure.
Significant impact of amount of PCR input templates on various PCR-based DNA methylation analysis and countermeasure.
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PCR输入模板量对各种基于PCR的DNA甲基化分析的显着影响及对策
DOI：
10.18632/oncotarget.10906
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发表时间：
2016-08-30
期刊：
影响因子：
--
通讯作者：
Deng D
中科院分区：
文献类型：
--
作者：
Liu Z;Zhou J;Gu L;Deng D
CpG岛的甲基化变化可以使用基于PCR的测定来确定。然而，输入模板（TAIT）的量对DNA甲基化分析的确切影响以前尚未被认识到。使用COL 2A 1基因作为输入参考，计算两个代表性基因GFRA 1和P16在甲基化阳性和阴性检测的人体组织之间的TAIT差异。结果显示，GFRA 1甲基化阳性的冻存标本（n = 332）TAIT显著高于甲基化阴性的冻存标本（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TAIT相关效应。进一步研究表明，当甲基化阳性检测的甲基化GFRA 1比例的临界值设定为1.6%时，成功MethyLight PCR反应的最小TAIT应≥ 9.4 ng（CtCOL 2A 1 ≤ 29.3）。在甲基化无信息的冷冻样本（n = 94; CtCOL 2A 1> 29.3）的TAIT增加到最小TAIT以上后，GFRA 1的甲基化阳性率分别从72.3%增加到95.7%，P16的甲基化阳性率从26.6%增加到54.3%（Ps < 0.001）。在FFPE样品中观察到类似的结果。总之，TAIT严重影响各种基于PCR的DNA甲基化分析的结果。表征靶CpG岛的最小TAIT对于避免假阴性结果至关重要。
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.