采用荧光定量RT-PCR法检测超极化激活环核苷酸门控阳离子通道

Qualification of hyperpolarization-activated cyclic nucleotide-gated cation channel mRNA using the fluorogenic quantitative RT-PCR method

目的建立用SYBR Green荧光染料检测超极化激活环核苷酸门控阳离子通道(HCN4)的实时定量RT-PCR方法。方法提取窦房结细胞的总RNA,反转录成cDNA后,应用RT-PCR法检测HCN4的表达,通过琼脂糖凝胶回收PCR产物并构建重组质粒,用梯度稀释的质粒模板建立荧光定量RT-PCR法检测HCN4的标准曲线,并检测心脏组织中HCN4的表达水平。结果成功构建了质粒重组子,并建立用SYBR Green荧光染料在RNA水平检测HCN4通道的标准曲线,相关系数为0.997重复6次实验组内和组间变异系数均<1.0%。心脏组织中窦房结HCN4表达量最高,心室表达量最低。结论基于SYBR Green荧光染料建立的定量RT-PCR体系敏感性高,线性范围广,重复性好,可快速准确地检测HCN4的表达。

AIM： To establish a fluorogenic quantitative RT-PCR （RTqPCR） method with SYBR Green dye for detecting the expression of hyperpolarization-activated cyclic nucleotide-gated cation channel （HCN4） mRNA. METHODS： Total RNA was extracted from the cardiac sinoatrial node tissue. After reverse transcription, the HCN4 gene was detected using RT-PCR method. PCR products were then gelpurified and subeloned into the PGEM-T Easy vector to construct the recombinant plasmid. The standard curve of the HCN4 gene was established by RTqPCR and used to detect the expression of HCN4 gene in different heart tissues. RESULTS： We successfully constructed the recombinant plasmid and established the standard curve to detect the expression of the HCN4 gene. There was good statistical linear relationship with the regression value 0. 997. In each experiment, the intra- and inter-assay coefficients of variation were 〈 1.0%. HCN4 expression was found at the highest level in sinoatrial node and the lowest level in ventricle. CONCLUSION： Real-time RT-PCR based on SYBRGreen dye has been developed with high specificity and sensitivity, which can be well repeated. This method is rapid and can be widely applied for HCN4 gene detection.