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目的 :建立 PCR辅助转录滴定系统 (PATTY)定量检测 AFP m RNA的方法。方法 :采用 RT- PCR定点替代突变及体外转录技术合成单碱基突变 AFP m RNA内竞争模板 ,以竞争性 RT- PCR定量检测各肝癌细胞株 AFP m RNA。结果 :成功制备引入 Hind 酶切位点的单碱基突变 AFP m RNA内竞争模板 ,建立 PATTY定量检测 AFP m RNA的方法 ;检测 1μgHep G2 和 Huh7两株肝癌细胞总 RNA中 AFP m RNA含量皆为 10 1 0 copy/ μg,PL C/ PRF/ 5细胞株为 10 7copy/ μg。 结论 :PATTY检测 AFP m RNA能同步监控待检靶 m RNA逆转录过程 ,扩增效率不受 PCR诸因素干扰 ,检测结果可靠 ,适合微量循环肝癌细胞相关 AFP m RNA的定量检测研究。
Objective: To establish a method for the quantitative detection of AFP m RNA by PCR-assisted transcriptional titration system (PATTY). Methods: A single base mutation AFP m RNA internal competitive template was synthesized by RT-PCR site-directed mutagenesis and in vitro transcription technology. AFP m RNA was detected by competitive RT-PCR. Results: A single-base mutant AFP m RNA competition template was successfully constructed and a PATTY method was established for the quantitative detection of AFP m RNA. The detection of AFP m RNA content in total RNA of 1 μg Hep G2 and Huh7 hepatoma cells was 10 1 0 copy / μg, PL C / PRF / 5 cell line was 10 7copy / μg. Conclusion: The PATTY detection of AFP m RNA can synchronously monitor the reverse transcript of target m RNA to be detected. The amplification efficiency is not disturbed by PCR and the results are reliable. It is suitable for the quantitative detection of AFP m RNA in microcirculation hepatocellular carcinoma cells.