实时荧光半定量MSP检测肺癌组织CDH1甲基化水平实验条件的探讨

Exploration with the Optimized Reaction Conditions of Real-Time Semiquantitative MSP for CDH1 Promoter Methylation in Lung Cancer Tissues

目的探讨实时荧光半定量MSP检测肺癌组织中CDH1DNA甲基化水平的最适实验条件。方法通过改变实时荧光半定量MSP反应条件,探讨MSP反应体系最适浓度和反应条件。结果检测CDH1DNA甲基化实时荧光半定量MSP反应体系为dATP,dCTP和dGTP200μmol/L,dUTP400μmol/L,MgCl23.0mmol/L,引物600nmol/L,热启动TaqDNA聚合酶0.04U/μl,模板3μl,DMSO2.5,1×SYBGREEN染料,1×反应缓冲液,总体系25μl。反应条件为95℃预变性12min,95℃15s、退火温度1min(其中62℃2个循环、60℃3个循环和58℃45个循环)、69℃10s,循环次数50。结论改进和建立了CDH1DNA甲基化水平的相对定量检测方法。

Objective To explore the optimized reaction conditions of semiquantitative fluorescence-based real-time MSP to detect CDH1 aberrant methylation of the lung cancer tissues. Methods Explored the optimized reaction conditions by changing those of semiquantitative fluorescence-based real-time MSP. Results The optimized reaction mixtures contained the forward and reverse primers at 600 nmol/L each; 200 μmol/L each of dATP,dCTP and dGTP ,400μmol/L dUTP, 3.0 mmol/L MgCl2,2.5 %DMSO, 1 × SYBGREEN I , 1 X×PCR buffer,hot start Taq DNA polymerase 0.04 U/μl and templates 3 μl. The total reaction volume was 25 μl. The semiquantitative real-time MSP was performed under the following conditions：95℃ for 12 minutes, followed by 2 cycles of 95 ℃ for 15 seconds, 62 ℃ for 1 minute and 69 ℃ for 10 seconds, 3 cycles of 95℃ for 15 seconds,60 ℃ for 1 minute and 69 ℃ for 10 seconds,and 45 cycles of 95 ℃ for 15 seconds,58 ℃ for 1 minute and 69℃ for 10 seconds. Conclusion The relative quantitative method for CDH1 aberrant methylation of lung tissues has been established.