大鼠bFGF基因荧光定量PCR检测方法的建立

Establishment of the method for detecting rat bFGF mRNA with fluorescence quantitative ploymerase chain reaction

目的建立用荧光定量PCR方法检测大鼠bFGF基因mRNA水平。方法以Trizol一步法提取新鲜骨组织总RNA，以Oligo（dt）18为引物逆转录产生cDNA并进行扩增，以T-A克隆法将纯化的目的片断与PGEM—T Easy载体连接成重组质粒并转化E．coli DH5α。采用碱裂解法提取重组质粒，经蓝白筛选、酶切、测序鉴定后，根据标准品建立标准曲线，由软件自动计算出待测样本中靶基因mRNA准确含量，并以靶基因和内参GAPDH mRNA含量的比值作为评价靶基因表达水平的指标。结果由PGEM-T Easy—bFGF所构建的标准曲线线性关系良好（反应体系中含1×10^2～1×10^7拷贝大鼠bFGF基因分子时，扩增反应α值与拷贝数的对数成线性关系）、灵敏度高、特异性强、准确可靠。批内和批间重复性测定的变异系数分别为1．03％～4、59％以及2．24％～6．46％。结论成功建立了用实时荧光定量PCR检测大鼠bFGF基因表达量的方法。

Objective The method for detecting rat bFGF mRNA expression with fluorescence quantitative polymerase chain reaction was developed. Methods Total RNA was isolated form fresh bone tissue sample using Trizol one step method and reverse transcribed to cDNA using Oligo（dt）18 as primer. The target fragment of bFGF cDNA was amplified and then was linked with PGEM-T easy vector to construct reeombined plasmid with T-A clone method. Recombined plasmids transformated to E. coli DH5α were extracted with alkaline lysis method. Target plasmids in white colonies selected by ampicillin screening were linearrizated by EcoR I restrictive enzyme and had their specificity identified by DNA sequencing. According to the standard curves created by plasmid DNA, the expression level of target genes in samples have been determined using software. The results were presented as the ratios of target genes＇ mRNA to GAPDH＇ mRNA. Results The standard curve made by PGEM-T easy bFGF had good linear dependence, and it was sensitive and specific. The coefficient of variation values for both intraexperimental and inter-experimental reproducibility ranged from 1.03% to 4.09% and 2.24% to 4.46%, respectively. Conclusion The method for detecting rat bFGF mRNA expression with fluorescence quantitative polymerase chain reaction was developed successfully.