代谢综合征相关新基因MSRG实时荧光PCR检测方法的建立

Establishment of a real-time PCR method for quantitative detection of a novel metabolic syndrome related gene (MSRG)

目的建立测定MSRG mRNA表达量的实时荧光定量PCR检测方法,为新基因的功能研究奠定基础。方法根据MSRGcDNA序列设计荧光PCR适用的引物和探针,构建质粒标准品,建立标准曲线用于荧光PCR相对定量,检测荧光PCR方法的灵敏性、特异性和重复性。荧光PCR检测不同浓度葡萄糖[5.6mmol/L(G5.6)、22mmol/L(G22)、33.3mmol/L(G33.3)]刺激人肝癌细胞株HepG2细胞MSRG的表达并与半定量RT-PCR方法进行比较。结果建立了MSRG mRNA表达的实时荧光PCR检测方法。荧光PCR检测显示G33.3、G22和G5.6组HepG2细胞MSRG表达均有显著差异,半定量RT-PCR方法检测G33.3组MSRG表达显著增加,但不能检测出G22和G5.6组间有差异。结论本实验建立的MSRG mRNA表达实时荧光PCR检测方法灵敏性、特异性和重复性较好,为MSRG功能的研究提供了一种重要手段。

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. Method. Specific primers and probes were designed for real-time PCR according to the MSRG cDNA sequence. The plasmid standard preparations were constructed by TA 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. 3rnmol/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 GS. 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.