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

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 gel-purified and subcloned 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.