摘要:
首次建立以GAPDH为内参,同时检测鸭RIG-I、MDA5、IFN-α和IFN-γ等4种mRNA相对表达量的SYBR GreenⅠ实时荧光定量PCR方法(RT-FQ-PCR)。以目的基因的克隆质粒为标准品,建立标准曲线,并进行熔解曲线、重复性及稳定性分析。该方法 Ct值线性范围为12.0~32.0;目的基因(RIG-I、MDA5、IFN-α、IFN-γ)和内参基因(GAPDH)的扩增效率均在95%~105%,其相关系数均在0.99以上;扩增产物的熔解曲线都只出现1个特异性单峰,无引物二聚体或其他非特异性产物生成,RIG-I、MDA5、IFN-α、IFN-γ和GAPDH的Tm值依次为(81.9±0.33)、(81.9±0.32)、(93.6±0.23)、(81.8±0.28)、(87.8±0.24)℃,其敏感度均达到100copies·μL-1,重复性和稳定性好,为从mRNA水平上深入研究RIG-I样受体、干扰素与鸭机体免疫功能相关性奠定了基础。
Abstract:
This study established detection of duck RIG-I,MDA5,IFN-αand IFN-γ mRNA relative expression levels synchronous of SYBR GreenⅠreal-time quantitative PCR for the first time.GAPDH was used as an internal reference.Cloning plasmids of objective genes were used as the standards for establishing standard curves for analysis of the melting curve,repeatability and stability.The linear range of Ct values of the method was 12.0to32.0.The amplification efficiencies of target genes(RIG-I,MDA5,IFN-α,IFN-γ)and the reference gene(GAPDH)were from 95% to 105%,and the correlation coefficients were above 0.99.The melting curve of amplification product only appeared a specific single peak,and there was no primer dimer or other non-specific product formation.Tm values of RIG-I,MDA5,IFN-α,IFN-γand GAPDH were(81.9±0.33),(81.9±0.32),(93.6±0.23),(81.8±0.28)and(87.8±0.24)℃,respectively.The sensitivity reached 100copies·μL-1,and repeatability and stability were good.This method would lay the foundation for further study of correlation between duck RIG-I like receptor,interferonand immune function from the mRNA levels.
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