摘要
目的 :建立可定量测定人端粒酶催化亚单位 (hTERT)mRNA的实时荧光反转录PCR方法。方法 :用Trizol 试剂从HepG2细胞中分离总RNA ,然后反转录为cDNA。利用一对基因特异引物和一条TaqManMGB探针 ,以实时荧光PCR定量hTERT的cDNA。同时定量测定人 β 肌动蛋白 (hBA)的mRNA作为内对照。用梯度稀释的克隆的人 β 肌动蛋白基因制作标准曲线。结果 :标准曲线的相关系数为 1.0 0。实时荧光反转录PCR方法的平均变异系数为 7.1%。HepG2细胞hTERTmRNA与hBAmRNA的比值为 (1.3± 0 .3)× 10 -4。结论 :建立了可定量测定人端粒酶催化亚单位 (hTERT)mRNA的实时荧光反转录PCR方法。
AIM: To establish a real-time fluorescent RT-PCR assay to quantify human telomerase reverse transcriptase (hTERT) mRNA. METHODS: Total cellular RNA was isolated from HepG2 cells using Trizol reagent, which was then reverse-transcribed into cDNA. By using a pair of gene-specific primers and a TaqMan MGB probe, cDNA of hTERT was quantified with real-time fluorescent PCR. Human β-actin (hBA) mRNA was quantified at the same time as an endogenous control. Serial dilutions of cloned human β-actin gene were used to construct a standard curve. RESULTS: The correlation coefficient of the standard curve was 1.00. The mean coefficient of variation of the assay was 7.1%. The ratio of hTERT mRNA to hBA mRNA of HepG2 cells was (1.3±0.3)×10 -4. CONCLUSION: A real-time fluorescent RT-PCR assay to quantify hTERT mRNA was established, which will facilitate the study on the biological function of telomerase.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第5期618-620,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
陕西省自然科学基金资助项目 (No .2 0 0 3C2 1 7)
