人mGluR1基因mRNA实时荧光定量PCR方法的建立

Establishment of real-time PCR assay for quantitative analysis of mGluR1 mRNA in human

目的建立代谢型谷氨酸受体1(mGluR1)基因mRNA表达水平的Taq Manreal-time PCR检测方法。方法以β-actin为内参基因,根据GenBank中人mGluR1及β-actin基因序列,分别设计了两套特异性引物和TaqMan探针,接着对反应的退火温度、引物浓度、探针浓度、Mg2+浓度进行优化,然后以优化的条件建立相对定量标准曲线,并对该方法的稳定性进行分析。结果mGluR1及β-actin基因的real-time PCR扩增效率分别为99.7%和100.0%;相对定量标准曲线的CT值线性范围分别为8.1～30.9和11.9～32.1,相关系数分别为0.999及1.000;批内及批间变异系数<6.4%。结论本研究所建立的针对mGluR1 mRNA表达水平的Taqman real-time PCR检测方法具有扩增效率高、稳定性好等特点,为进一步探索mGluR1的功能及其mRNA表达水平的变化和各种疾病发生、发展的相关性提供了方法学基础。

Objective To establish a TaqMan real-time PCR assay for quantitative analysis of human mGluR1 mRNA expression. Methods According to the sequences of mGluR1 and β-actin genes（ as a reference control ） in GenBank, two sets of primers and TaqMan probes were designed. Subsequently, PCR conditions, including annealing temperature, concentration of primers, probes and Mg^2＋ were optimized. A relative quantitative standard curve was established and repeatability of the assay was also evaluated. Resalts PCR efficiency of mGluR1 and β-actin was 99.7% and 100. 0% respectively, the linear range of standard curve for relative quantification was 8. 1 - 30. 9 and 11.9 - 32.1, the correlation coefficient was 0. 999 and 1. 000 respectively. Intra-assay and inter assay coeffeciency variation for both genes were less than 6. 4 %. Conclusion The real-time PCR for mGluR1 established in this study have advantages of high PCR efficiency and good consistency, and will provide useful methodological basis for understanding functions of mGluR1 and clarifying relationship between mRNA expression level and occurrence, development of certain disease.