Establishment of a real-time PCR method for quantitative detection of a novel metabolic syndrome related gene (MSRG) / 解放军医学杂志

ABSTRACT

Objective To establish a Taqman real-time PCR assay for quantitative detection of the expression of metabolic syndrome related gene (MSRG) mRNA, and to study the function of a new gene. Methods Specific primers and probes were designed for real-time PCR according to the MSRG cDNA sequence. The plasmid standard preparations were constructed by T-A clone, and serial 10-fold dilutions of the extracted plasmid standard preparations were prepared for plotting the standard curve which was used for relative quantification of real-time PCR. The sensitivity, specificity and reproducibility of real-time PCR assay were detected. To compare with the semi-quantitative RT-PCR, the expression levels of MSRG were measured by real-time PCR and semi-quantitative RT-PCR, respectively, in HepG2 cells which were incubated with glucose in different concentrations [5.6mmol/L (G5.6), 22mmol/L (G22) and 33.3mmol/L(G33.3)]. Results An effective real-time PCR assay was established for detection of MSRG mRNA expression levels. Significant differences existed in MSRG expression in HepG2 cells between the G33.3, G22 and G5.6 groups detected by real-time PCR assay. The expression levels of MSRG in HepG2 cells increased significantly in G33.3 group, whereas no significant difference on the expression level of MRSG mRNA was found between G22 and G5.6 groups when semi-quantitative RT-PCR was used for detection. It suggested that the real-time PCR assay was more sensitive and even more precise than that of semi-quantitative RT-PCR. Conclusion The real-time PCR assay was a sensitive, specific, quantitative, and reproducible tool for studying the function of MSRG at the mRNA expression levels of gene.