獐茅AlHAK1 Real-time PCR定量方法的建立

Establishment of Real-time PCR Assay for Quantitative Analysis of AlHAK1

以β-actin为内参基因,根据已克隆的AlHAK1及β-actin碱基序列,分别设计了实时定量特异性引物,优化了反应的退火温度与引物浓度,并以优化的条件建立了相对定量标准曲线,同时对该方法的稳定性进行了评价。结果表明AlHAK1及β-actin基因的real-time PCR扩增效率分别为1.09和1.04,线性相关系数分别为0.996和0.997,批内及批间变异系数<5%。所建立的AlHAK1real-time PCR定量方法操作简便、定量准确、重复性好,为进一步探索AlHAK1的功能、mRNA表达水平的变化及转AlHAK1农作物的检测奠定了方法学基础。

According to the sequences of AlHAK1 and β-actin （as a reference control）, two sets of primers were designed. Subsequently, PCR conditions, including annealing temperature and concentration of primers were optimized. Relative quantitative standard curves were established and the repeatability of the assay was also evaluated. The results show that PCR efficiency of AlHAK1 and β-actinwas 1.09 and 1.04, respectively. The correlation coefficient was 0.996 and 0.997, respectively. Intra-assay and inter-assay eoefficieney variation for both genes were less than 5%. The real-time PCR for AlHAK1 established in this study is simple, precise and repeatable for relative quantifying gene, and will provide useful methodological basis for understanding functions of AlHAK1, clarifying relationship between mRNA expression levels and detecting the expression level of transgenic crops.