PCR辅助转录滴定系统定量检测AFP mRNA方法的建立

A method for quantitation of AFP mRNA by PCR aided transcript titration assay

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 develop a PCR aided transcript titration assay(PATTY) system for quantitation of AFP mRNA . Methods:RT PCR oligonucleotide overlap extension and in vitro transcription were performed with specific AFP primers to construct the mutant fragment of competitor AFP mRNA, AFP mRNAs in hepatoma cell lines were quantitatively analyzed by competitive RT PCR. Results:The mutant fragment of competitor AFP mRNA with a new introduced restriction enzyme site of Hind Ⅲ was constructed and then PATTY system for quantitation of AFP mRNA was developed successfully. AFP mRNA levels in both HepG 2 and Huh7 cell lines were 10 10 copy/μg RNA, in contrast to 10 7 copy/μg RNA in PLC/PRF/5 cell line. Conclusion:The result of quantitating AFP mRNA by this PATTY system is reliable, the competitor AFP mRNA template can supervise the processes of reverse transcription and PCR amplification for detection of target mRNA synchronously. The system may quantitate circulating hepatocellular carcinoma cells associated AFP mRNA . [