实验动物科学 ›› 2015, Vol. 32 ›› Issue (03): 33-37.

• 研究 论著 • 上一篇    下一篇

猕猴不同组织器官中黄嘌呤脱氢酶 /氧化酶基因的mRNA 表达的半定量 RT - PCR 检测方法的建立

  

  1. (1. 中国医学科学院/北京协和医学院 医学生物学研究所,昆明 650118) ( 2. 云南省中医学院,昆明 650200)
  • 出版日期:2015-06-29 发布日期:2017-04-13

Establishment of a SEMI-QUANTITATIVE RT-PCR Method for the Detection of XDH /XO mRNA Expression in Difference Organ Tissue

  • Online:2015-06-29 Published:2017-04-13

摘要: 摘要: 目的 建立半定量 RT-PCR 检测方法检测猕猴体内肝脏组织、上皮组织、睾丸组织、心肌组织,肾脏组织中黄嘌呤氧化脱氢酶( XDH /XO) 的 mRNA 的表达。方法 分别提取健康猕猴新鲜肝脏组织、上皮组织、睾丸组织、心肌组织,肾脏组织中总 RNA,用 RT-PCR 二步法进行 XDH /XO 基因片段扩增,内参基因选用管家基因 GAPDH,对获得的目的片段进行序列测定以鉴定其特异性,在凝胶影像分析仪 Quantity one 软件得出其电泳条带的灰度值( IOD) ,计算目的基因与内参基因比值得到猕猴不同组织 XDH/XO 的 mRNA 的相对表达量。结果 建立了半定量 RT-PCR 检测方法,猕猴不同组织 XDH /XO 的 mRNA 表达在肝脏组织中表达水平最高,其次为心肌,肾脏,睾丸,皮肤。结论 本研究所建立的半定量 RT-PCR 检测方法可应用于检测猕猴各组织器官组中的 XDH /XO 的mRNA 的表达差异。

关键词: 黄嘌呤氧化/脱氢酶, 猕猴, 基因表达, RT-PCR

Abstract: Abstract: Objective To established of a semi-quantitative RT-PCR method for the detection of xanthine dehydrogenase /oxidase( XDH /XO) mRNA expression from tissue of the cardiac muscle,kidney,testicle,skin,liver of rhesus monkey. Method Total RNA was extracted from the cardiac muscle,kidney,testicle,skin,liver tissue respectively for amplification of XDH /XO gene fragment by two-step RT-PCR. GAPDH is as a ‘reference’genes to normalize RT-qPCR data. The specificity of sequence of amplified nucleotide acids were identified by gel electrophoresis and sequencing. The gray scale rate ( IOD ) of electrophoresis strip were obtained from quantity One of GelDocTM XR gel image analyzer. Result A semi-quantitative RT-PCR method have been established for the detection of XDH /XO mRNA in different tissues of the rhesus monkey. Quantitative analysis demonstrated highest expression of XDH /XO in liver,and then cardiac muscle,kidney,testicle,skin. Conclusion The semiquantitative RT-PCR method will be apply to detect XDH /XO mRNA expression in difference organ tissue.

Key words: XDH /XO, Macaca mulatta, expression of gene, RT-PCR

中图分类号: