家兔GAPDH基因实时荧光定量RT-PCR方法的建立

根据GenBank登录的家兔GAPDH基因保守区域CDS序列设计1对引物,通过反应体系及反应条件优化,成功建立了检测家兔GAPDH基因的SYBR GreenⅠ荧光定量RT-PCR方法。结果表明,该方法检测GAPDH的最低拷贝数为32拷贝/μL,在较广的范围内(3.2×101~3.2×107拷贝/μL)有良好的线性关系(r=0.999);熔解曲线分析显示扩增产物的特异性单峰,其Tm为(87±0.2)℃;5个不同浓度标准品组内试验变异系数为1.67%~4.73%,组间试验变异系数为2.66%~8.74%。该方法具有快速、灵敏、高通量及可重复性强等优点,为GAPDH基因作为内参基因进行家兔功能基因与病原基因表达的定量分析提供了方法学基础。

Development of a Real-time RT-PCR Assay for Quantification ot Rabbit GAPDH Gene

According to the published GAPDH gene’s CDS sequence in GenBank, a pair of primers was designed and synthesized. Through optimization of react system and conditions, the method for detection of GAPDH gene of rabbit by SYBR Green Ⅰreal-time RT-PCR was established successfully. The results show that the lowest copy number for detection of GAPDH gene with this method is 32 copies/μL. and there is a good linear relationship in a wide range from 3.2×101 to 3.2×107 copies/μL (r=0.999). The coefficient of variation (CV) of 5 different concentration of positive plasmids is 1.67% to 4.73% and 2.66% to 8.74% in intra-assay and in inter-assay respectively. The method established in this paper has the advantages of rapidity,high sensibility, high throughput and good repeatability, which provides a methodological basis for quantitative analysis on function gene of rabbit and the expression of some disease gene in rabbit when GAPDH gene is taken as a reference gene.