Taqman技术检测肿瘤细胞中端粒酶逆转录酶信使核糖核酸及表达的意义  

Quantitation of human telomerase reverse transcriptase mRNA with Taqman technique

作　　者：李一荣[1] 陈凤花[1] 王琳[2] 王利君[1] 胡丽华[3] 吴健民[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院检验科免疫学研究所,武汉430022 [2]武汉大学生命科学院 [3]华中科技大学同济医学院附属协和医院输血科,武汉430022

出　　处：《中华检验医学杂志》2004年第1期20-22,共3页Chinese Journal of Laboratory Medicine

基　　金：湖北省科技攻关项目 (2 0 0 2AA3 0 4B10 )

摘　　要：目的　用定量Taqman技术检测人端粒酶逆转录酶 (hTERT)信使核糖核酸 (mRNA)以及在肿瘤细胞中表达的意义。方法　在Trizol提取总RNA后 ,将mRNA逆转录成互补脱氧核糖核酸 ,用Taqman技术定量检测hTERTmRNA ,并与经典逆转录聚合酶链反应 (RT PCR)方法比较。结果　急性髓细胞白血病患者hTERTmRNA表达水平为 (5 2 9～ 7 3)× 10 4拷贝 / μl,平均 3 5× 10 3 拷贝 / μl,其阳性率 (90 3% )高于健康体检者 (5 6 % ,χ2 =2 9 9,P <0 0 5 ) ,但与经典RT PCR检出阳性率比较 ,差异无显著意义 (χ2 =1 3,P >0 0 5 )。Taqman技术灵敏度为 1 5～ 3拷贝 / μl,特异性为10 0 % ;在 (5 0～ 5 )× 10 8拷贝 / μl之间有良好的线性关系 ,相关系数为 10 0 % ;5 0和 5 0 0 0拷贝 / μl的标本的内变异系数 (CV) 1 7%和 2 0 % ,日间CV则分别为 3 7%和 2 9%。结论　Taqman技术是一种快速有效、灵敏度高、特异性良好的定量检测hTERTmRNA的方法。Objective To establish a Taqman technique for quantitating human telomerase reverse transcriptase mRNA and study its expression in tumour cells . Methods Total RNA was extracted with Trizol and mRNA was transcribed reversely into cDNA. Taqman technique was used to quantitate hTERT mRNA. The method was reviewed and compare to the result of classical RT-PCR. Results In patients with AML,the expression level of hTERT mRNA ranges from 52.9 to 7.3×10 4 copies/μl,the mean value was 3.5×10 3 copies/μl. The positive rate was 90.3%,whereas in healthy people it was 5.6% and there was significant difference in positive rate of the two group(χ 2=29.9, P <0.05). No significant difference in positive rate detected by Taqman technique and classical RT-PCR (χ 2=1.3, P >0.05). The sensitivity was from 1.5 to 3 copies/μl and the specificity was 100%. A good linearity was found from 50 to 5×10 8 copies /μl and the correlation coeffecient was 1. When the input of human telomerase reverse transcriptase was 50 copies/μl, the interexperimental coeffecient of variation and the coeffecient of variation between days were 1.7% and 3.7% respectively,whereas the input was 5000 copies/μl, they were 2.0% and 2.9% respectively. Conclusion Taqman technique is a rapid and sensitive and good specific method for quantitating human telomerase reverse transcriptase mRNA.

关 键 词：TAQMAN技术 检测 肿瘤细胞 端粒酶逆转录酶 信使核糖核酸 表达 

分 类 号：R730.4[医药卫生—肿瘤]