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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 detect the expression of human telomerase reverse transcriptase (hTERT) messenger RNA (mRNA) and its expression in tumor cells by quantitative Taqman technique. Methods Reverse transcription of mRNA into complementary deoxyribonucleic acid (DNA) was performed after total RNA was extracted from Trizol. The hTERT mRNA was quantitatively determined by Taqman technique and compared with that of classical reverse transcriptase polymerase chain reaction (RT PCR). Results The expression of hTERT mRNA in patients with acute myeloid leukemia was (529 ~ 7 3) × 10 4 copies / μl with an average of 35 × 10 3 copies / μl. The positive rate of hTERT mRNA expression was 90 3% 6%, χ2 = 299, P <0 05), but there was no significant difference (P> 0.05) with the positive rate of classical RT PCR. The Taqman technique has a sensitivity of 15-3 copies / μl and a specificity of 10%. There is a good linear relationship between (5 0-5) × 10 8 copies / μl with a correlation coefficient of 10 0% The intra-CV (CV) values of 1700 and 20% of the 5 0 0 0 copies / μl were 37% and 29%, respectively. Conclusion Taqman technique is a rapid, effective, sensitive and specific method for the quantitative detection of hTERT mRNA.