鸭组织实时荧光定量PCR内参基因表达稳定性的筛选研究

Screening of Reference Gene Expression Stability for Real-time Quantitative PCR in Duck Tissues

为研究不同内参基因在鸭组织中表达的稳定性,试验采用实时荧光定量PCR技术(qRT-PCR)检测了3磷酸甘油醛脱氢酶(GAPDH)、β肌动蛋白(β-actin)、18S核糖体RNA(18S rRNA)和16 S核糖体RNA(16S rRNA)4个常见内参基因在21和42日龄樱桃谷鸭肝脏、回肠、胸腺、脾脏、腿肌、下丘脑、心脏、十二指肠、脂肪和胸腺组织中的表达情况,使用geNorm软件对各内参基因表达的稳定性进行分析。结果显示:4个内参基因在21日龄各组织的Ct值高低顺序依次为:18S rRNA>16S rRNA>GAPDH>β-actin,在42日龄各组织的Ct值高低顺序依次为:18S rRNA>β-actin>GAPDH>16S rRNA;4个内参基因在不同组织的稳定性有所不同,表明内参基因稳定性具有组织特异性。研究提示:对鸭的多组织进行基因表达水平定量时,16S rRNA和18S rRNA联合使用的效果最佳,而对单一组织进行基因表达水平定量时,则需选择该组织中稳定性最好的内参基因。

To study the expression stability of different reference genes in duck tissues, real-time quantitative PCR(qRTPCR) was used to detect glyceraldehyde-3-phosphate dehydrogenase(GAPDH), β-actin, 18S rRNA and 16S rRNA in liver, ileum, thymus, spleen, leg muscle, hypothalamus, heart, duodenum, fat and thymus of Cherry Valley ducks at 21 and 42 days of age.The geNorm software was used to analyze the stability of gene expression. The results showed that the order of Ct values of four reference genes in each tissue at 21 days of age was 18S rRNA>16S rRNA>GAPDH>β-actin, whereas those in each tissue at 42 days of age was 18S rRNA>β-actin>GAPDH>16S rRNA. The stability of four reference genes in different tissues was different,indicating that the stability of internal reference genes was tissue-specific. It′s demonstrated that the combination of 16S rRNA and 18S rRNA was the best for quantitative analysis of gene expression level in multiple tissues of duck, and the internal reference gene with the best stability in single tissue should be selected for quantitative analysis of gene expression level.