TaqMan实时荧光定量RT-PCR检测外周血CK19 mRNA

目的:应用TaqMan实时荧光定量RT-PCR技术,建立定量检测CK19 mRNA的方法. 方法:采用RT-PCR从胃癌细胞中克隆CK19片段(230 bp),装入pMD 18-T Simple载体.纯化质粒,制备荧光定量PCR标准品.应用LightCycle荧光定量PCR仪检测标准品、30例正常人外周血和5例肿瘤组织CK19 mRNA. 结果:PCR扩增及测序均证实CK19 cDNA片段重组到pMD 18-T载体上,建立了稳定的检测CK19 mRNA的标准,即设定CT值在35个循环之内,检测结果低于100个拷贝数量级为阴性标本.结论:采用TaqMan实时荧光定量RT-PCR技术检测外周血CK19 mRNA是一种稳定可靠的方法.

TaqMan real time quantitative RT-PCR in detection of peripheral blood CK19 mRNA

Objective:To establish a quantitative method for determination of CK19 mRNA with TaqMan real time quantitative RT-PCR.Methods: A 230 bp fragment of CK19 mRNA was amplified from the total RNA of gastric cancer cells using RT-PCR methods and was introduced into pMD 18-T Simple vector.The plasmid was purified and the fluorescent standard PCR product was prepared.The expression levels of CK19 mRNA in standard PCR product,5 tumor tissue specimens and 30 healthy subjects were observed.Results: A 230 bp fragment of CK19 mRNA was successfully cloned into the pMD 18-T Simple vector and was verified by sequence analysis.A stable standard for detection of CK19 mRNA was established,that is,when C_(T) was set within 35 cycles,negative specimen was defined when the result was lower than 100 copies.Conclusion: TaqMan real time quantitative RT-PCR is stable and reliable in quantitative detection of CK19 mRNA in peripheral blood.