建立检测猕猴三磷酸腺苷结合盒转运蛋白G2的mRNA相对表达水平的RT-qPCR方法

Establishing a Real-time qPCR Method for Detecting the mRNA Level of ABCG2 in Macaque

目的本研究旨在建立一种实时荧光定量PCR方法,用于检测猕猴三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate-binding cassette transporter protein G2,ABCG2)mRNA的基因转录水平。方法使用NCBI上GenBank数据库猕猴(Macaca mulatta)的ABCG2核苷酸序列号NM_001032919.1及内参GAPDH核苷酸序列号NM_001195426.1,借助Primer premier 5.0软件设计PCR引物。提取猕猴新鲜肾组织的总RNA,并反转录合成cDNA。接着,利用PCR引物进行实时荧光定量PCR扩增,并根据反应体系中荧光的变化情况定量分析ABCG2的mRNA相对表达水平。结果PCR产物测序结果显示,扩增的ABCG2和GAPDH核苷酸序列与NCBI上猕猴的序列同源性分别为90.91%和91.14%。ABCG2和GAPDH的扩增效率均达到80%~120%,实时荧光定量PCR标准曲线的熔解曲线为单峰,R2接近1。结论本研究建立的检测猕猴ABCG2 mRNA实时荧光定量检测方法,为研究高尿酸血症的发病机制以及新药开发奠定基础。

Objective To establish a real-time quantitative PCR method for the detection of mRNA transcription levels of adenosine triphosphate-binding cassette transporter G2(ABCG2)in macaque(Macaca Mulatta).Method We used ABCG2 nucleotide sequence number NM_001032919.1 and internal reference GAPDH nucleotide sequence number NM_001195426.1 of macaque in GenBank database on NCBI.PCR primers were designed using Primer premier 5.0 software.We extracted total RNA from fresh kidney tissue of rhesus monkey and synthesized cDNA by reverse transcription.Then,PCR primers were used for real-time quantitative PCR amplification,and the relative expression level of ABCG2 mRNA was quantitatively analyzed according to the changes of fluorescence in the reaction system.Result The PCR sequences showed that the homology of the amplified ABCG2 and GAPDH sequences was 90.91%and 91.14%,respectively,with that of the NCBI Macaca mulatta sequences.The amplification efficiency of ABCG2 and GAPDH reached 80%-120%.The meltdown curve of the standard real-time quantitative PCR curve was unimodal,and R2 was close to 1.Conclusion This study established a real-time fluorescence quantitative detection method for ABCG2 mRNA in macaque,which laid a foundation for the study of the pathogenesis of hyperuricemia and the development of new drugs.