Abstract: :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.

Key words: macaque ( Macaca Mulatta ) , real-time quantitative PCR ( RT-qPCR ) , adenosine triphosphate-binding cassette transporter G2 (ABCG2) 

摘要： 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,R 2 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。

关键词: 猕猴, 实时荧光定量 PCR, 三磷酸腺苷结合盒转运蛋白 G2