双重荧光实时定量PCR法检测TNF-α mRNA表达水平

Establishment of dual fluorescent quantitative real-time PCR for detection of TNF-α

目的:建立单管检测肿瘤坏死因子-α(TNF-α)和内参GAPDH mRNA表达水平的双重荧光实时定量PCR方法。方法:脂多糖刺激小鼠巨噬细胞Ana-1后提取细胞RNA,经反转录合成c DNA,应用自行设计的不同荧光标记的TNF-α和GAPDH引物探针,经PCR扩增后,在FAM通道和CY5通道分别读取Ct值,同时应用基因测序技术对结果进行验证,并构建质粒标准品分析该方法的灵敏度和重复性。结果:双重荧光实时定量PCR法检测TNF-α的灵敏度达4×101拷贝/μL,检测线性范围可达6个数量级,批内和批间重复性好。结论:新方法消除了目的基因与内参的加样误差,节约成本,快速准确,适用于TNF-αmRNA的定量检测。

Objective： Establish a dual fluorescent quantitative real-time PCR for detection of TNF-α. Methods： The RNA was isolated from Ana-1 cell line after inducing by lipopolysaccharide. Primers and probes were designed according to mice TNF-α and GAPDH gene. The threshold cycles were reading through channel FAM and channel CY5, respectively. After verifying the amplification results by gene sequencing, the standard plasmid was constructed for analyzing the sensitivity and repeatability of the method. Results： The analytical sensitivities of the dual real-time PCR assay for TNF-α and GAPDH reached to 4 × 10^1 copies/μL. No cross-reaction was observed during the reaction. The linear range and the intra-assay coefficient of variation were 4 × 10^1-4 × 10^6copies/μL and 〈 3.53%. Conclusion： The new method can eliminate the errors caused by adding samples. It is a simple, economic and suitable method for assaying ex- pression of target genes.