摘要
目的:构建增殖诱导配体(APRIL)基因的重组质粒的标准品。方法:从肺癌的组织标本中抽提总RNA逆转录合成cDNA,以cDNA为模板扩增目的片段,从胶中回收目的片段,再将回收的目的片段与pGEM-TEasy载体连接并转化宿主菌DH-5α,分别用菌落PCR及测序两种方法证实目的片段已成功转染细菌。抽提重组质粒DNA用核酸分析仪测浓度后换算成copies/ml。结果:成功构建了APRIL实时荧光定量标准品的线性范围105～1012copies/ml,批内与批间CV分别为1.34%～3.07%和7.24%～11.41%。结论:该法制备的质粒标准品具有较好的灵敏度和重复性,满足实时荧光定量实验要求。
Objective：To construct the standard recombinant plasmids for APRIL gene and using for quantification.Methods：Total RNA was extracted from the tissues of lung cancer and then cDNA was synthesized by reverse transcription.Target sequence was amplified from cDNA and then the PCR product was Gel extracted and purified .Products of purification ligated with pGEM-T Easy vector, and transformed into competent DH-5α, Two methods of colony PCR and sequencing were used to verify the target fragments are transformed successfully.Plasmid DNA is extracted and cDNA plasmid concentrations are measured spectrophotometrically and then converted to copies/ml according the equation.Results：Construction of APRIL standard for real-time fluorescence quantitation is achieved successfully.The standard has wide detection range, and the coefficients of variation value for both intraassay and interassay reproducibility range from 1.34%-3.07% and 7.24%- 11.41%,respectively.Conclusions ：The plasmid standard has a better sensitivity and reproducibility. It can satisfy the need of experiments.
出处
《交通医学》
2007年第1期14-16,共3页
Medical Journal of Communications
关键词
增殖诱导配体
聚合酶链反应
质粒标准品
T-A克隆
A proliferation-inducing ligand
Nucleic acid amplification technique
plasmid standard
T-A clone
