检测锯齿状腺瘤中CDX2基因甲基化率的两种qPCR方法比较

Comparison between two methods of qPCR for detection of methylation status of CDX2 gene in serrated adenoma

目的应用两种方法检测锯齿状腺瘤中CDX2基因甲基化状态,比较这两种检测方法的差异。方法采用Taqman探针qPCR(MethyLight)和SYBR Green qPCR方法检测129例锯齿状腺瘤(包括61例SSA/P、68例TSA)和42例正常结直肠黏膜组织中CDX2基因CpG岛甲基化状态。结果 MethyLight方法:SSA/P组织中CDX2基因甲基化率(75.41%,46/61)高于对照组(53.38%,22/42),两者差异显著(P<0.05)。TSA组织中CDX2基因甲基化率(80.88%,55/68)高于对照组(53.38%,22/42),两者差异显著(P<0.05)。SYBR Green qPCR方法:SSA/P、TSA组和对照组的组织中CDX2基因均呈高甲基化状态,SSA/P组织中CDX2基因甲基化率(81.97%,50/61)高于对照组(73.81%,31/42),两者比较差异不显著(P>0.05);TSA组织中CDX2基因甲基化率(86.76%,59/68)高于对照组(73.81%,31/42),两者比较差异不显著(P>0.05);结论 MethyLight消除了引物二聚体和非特异性扩增对试验结果的影响,提高了结果的特异性和准确性,被证实是进行异常DNA甲基化状态检测的可靠方法。

Objective To use two methods to detect the methylation status of CDX2 gene in the serrated adenoma and compare the effect of the two methods. Methods The methylation status of CpG island of CDX2 gene promotor was detected in 129 eases of serrated adenomas, including 61 cases of SSA/P and 68 cases of TSA, and 42 cases of normal colorectal mucosa with both Taqman probe based qPCR technology （MethyLight） （method 1 ）and SYBR Green based qPCR technology （method 2）. Results The restdts of MethyLight showed that the rate of CDX2 gene methylation in SSA/P group （75.41%, 46/61） was higher than that in normal eolorectal mucosa （53.38%, 22/42）, and there was a statistically significant difference between SSA/P and controls （P 〈 0. 05 ）, and the rate of CDX2 gene methylation in TSA group （80. 88%, 55/68）was higher than the rate of CDX2 gene methylation in normal eolorectal mucosa （53.38%, 22/ 42） , and there was a statistically significant difference between SSA/P and controls （ P 〈 0. 05 ）. The results of SYBR Green qPCR showed that there was a high methylation rate of CDX2 gene in SSA/P, TSA and normal colorectal mucosa. The rate of CDX2 gene methylation in SSA/P （81.97%, 50/61 ） was higher than that in controls （73.81%, 31/42） with X2 test analysis, but there was no statistically significant difference （P 〉 O. 05 ）. The rate of CDX2 gene methylation in TSA （86.76%, 59/68） was higher than that in controls （73.81%, 31/42） with X2 test analysis, but there was statistically significant difference （P 〉 0. 05 ）. Conclusion MethyLight can effectively eliminate the effect of the primer - dimers and nonspecific amplication, and improves the specificity and accuracy of the results, which can be used to detect the the methylation status of abnormal DNA.