摘要
目的比较荧光定量PCR与半定量PCR检测猕猴体内肝脏组织、上皮组织、睾丸组织、心肌组织,肾脏组织中黄嘌呤脱氢酶氧化酶(XDH/XO)的mRNA的相对表达量的差异,为改进实验研究方法学提供参考依据。方法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,使用相同的引物序列及内参基因,分别用荧光定量PCR与半定量PCR进行相对定量检测,结果做比对分析。结果两种检测方法均能检测到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XDH/XO的mRNA表达,荧光定量PCR灵敏度可检测到肝脏组织0.4ng总RNA中的XDH/XO的mRNA表达,半定量PCR灵敏度可检测到肝脏组织15.625 ng总RNA中的XDH/XO的mRNA表达,荧光定量PCR较半定量灵敏度高39倍。两种方法检测结果均显示猕猴不同组织XDH/XO的mRNA表达在肝脏组织中表达水平最高,对于表达量较低的其他组织,荧光定量PCR与半定量PCR检测结果存在有一定差异。结论两种方法均可用于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的相对表达量,荧光实时定量PCR灵敏度较高于半定量PCR法。
Objective To analyze the differences between the semi-quantitative RT-PCR and real time quantitative fluorescence RT-PCR assays for detecting XDH / XO mRNA expression in various organ tissues of rhesus monkey,and provide useful reference in methodology of experimental studies. Total RNA was extracted from the myocardium,kidney,testis,skin, and liver tissues, respectively, for detecting XDH / XO mRNA expression in rhesus monkey by semiquantitative RT-PCR and real time quantitative fluorescence RT-PCR assays. The sensitivity and specificity of the twoassays were compared with each other using the same primer sequences and reference genes. Results The expression of XDH / XO mRNA in different organ tissues were detected by both the two PCR assays. The sensitivity of quantitative fluorescence real-time RT-PCR for the XDH / XO mRNA expression in the liver tissue was 39 times higher than that by semiquantitative RT-PCR. Conclusions Both the quantitative and semi-quantitative fluorescence RT-PCR assays can be used to detect the expression of XDH / XO mRNA in different organ tissues of rhesus monkey. The sensitivity of quantitative fluorescence real-time PCR assay is more sensitive than that of the semi-quantitative RT-PCR assay.
出处
《中国比较医学杂志》
CAS
北大核心
2015年第12期47-53,共7页
Chinese Journal of Comparative Medicine
