实时荧光定量反转录聚合酶链反应检测bcl-2基因mRNA表达

Real-time quantitative RT-PCR for the detection of bcl-2 mRNA expression

目的 建立实时荧光定量反转录聚合酶链反应 (reverse transcription- polymerase chain reac-tion,RT- PCR)法检测 bcl- 2基因 m RNA表达。方法 应用 T- A克隆技术构建含 bcl- 2基因的载体作为标准模板 ,采用荧光 Taq man方法及探针标记技术 ,建立实时荧光定量 RT- PCR方法 ,制备标准曲线 ,检测bcl- 2反义核酸治疗前后人类 Burkitt's淋巴瘤裸鼠模型中瘤细胞的 bcl- 2 m RNA表达水平变化 ,并与半定量 RT- PCR检测结果进行比较。结果 实时荧光定量 RT- PCR检测 bcl- 2反义核酸治疗组的瘤细胞 bcl- 2m RNA表达明显降低 ,而 bcl- 2无义核酸对照组及空白对照组的瘤细胞 bcl- 2 m RNA表达仍较高 ,且显示无义核酸对照组与空白对照组的 bcl- 2 m RNA表达水平相近。半定量 RT- PCR结果显示 bcl- 2出现类似的变化趋势。结论 所建立的实时荧光定量 RT- PCR用于检测 bcl- 2 m RNA表达 ,结果用拷贝数表示 ,比半定量 RT- PCR更灵敏。

Objective: To establish a real-time quantitative RT-PCR method for the detection of expression of bcl-2 mRNA. Methods: The vector containing bcl-2 gene as standard template was constructed with T-A cloning technique. The fluorogenic probe (i. e., Taq man probe) was used to establish a real-time RT-PCR. bcl-2 mRNA expression level in Burkitt's lymphoma pre- and post-treated with bcl-2 antisense phosphothioate oligodeoxynucleotides (AS-PS-ODN) was determined with real-time quantitative RT-PCR, also with semi-quantitative RT-PCR. Results: The expression of bcl-2 mRNA in Burkitt's lymphoma treated with bcl-2 AS-PS-ODN decreased significantly, and no changes of bcl-2 mRNA expression in group treated with nonsense ODN were noticed. The semi-quantitative RT-PCR results also demonstrated that bcl-2 level varied as detected with real-time fluorogenic quantitative RT-PCR, but less sensitive and accurate. Conclusion: Detection of bcl-2 mRNA expression with the fluorogenic real-time quantitative RT-PCR is more accurate and sensitive than semi-quantitive RT-PCR.