SYBR Green I-based real-time quantitative RT-PCR method for detecting mRNA level of type I collagen in rats

AIM: To establish SYBR Green I-based real-time quantitative RT-PCR method for detecting type I collagen mRNA in rats. METHODS: Type I collagen cDNA was amplified by RT-PCR method and cloned into pMD18-T vector. The recombinant plasmid was selected and identified by sequence analysis. It was then diluted pro rata as standard template. The mRNA expression of type I collagen in rats was detected using real-time quantitative RT-PCR method with fluorescent DNA dye SYBR Green I. RESULTS: The recombinant plasmid was established and confirmed as expected. The kinetics graph after real-time PCR detected even one molecule. The standard curve indicated the liner relationship between CT (cycle threshold) and template concentration. The correlation coefficient of external standard between the input copies and fluorescence intensity was 0.991. CONCLUSION: The established method for detecting type I collagen mRNA in rats is sensitive and specific. Real-time quantitative RT-PCR based on SYBR Green I is an excellent candidate as a standard detection method for large-scale application.