绵羊基因表达定量分析内参基因的筛选

The Selection of Reference Genes of qRT-PCR Analysis in Sheep

内参基因在基因表达定量分析中常用于修正基因的表达量,但不同条件下内参基因的表达并不稳定。本实验旨在选出在绵羊不同组织稳定表达的内参基因。选取6月龄广灵大尾羊的皮肤、肾周脂肪、肝脏、背部最长肌组织为实验样本,用实时荧光定量PCR方法分别测定内参基因GAPDH、18S rRNA、B2M、ACTβ、RPLPO、YWHAZ在绵羊各组织内的mRNA丰度,经GeNorm、NormFinder及BestKeeper综合分析内参基因的稳定性。结果表明:在绵羊背部最长肌和肾周脂肪中,3个软件都得出GAPDH表达最稳定;在肝脏中,3个软件都得出RPLPO表达最稳定;在皮肤中,GeNorm和NormFinder均得出18S rRNA表达最稳定,而GeNorm和BestKeeper均得出GAPDH表达最稳定,所以选取18S rRNA或GAPDH作为内参基因;比较4个组织中表达最稳定的基因时,GeNorm和NormFinder软件均得出RPLPO表达最稳定,而BestKeeper分析表明B2M基因稳定性最好,所以选取RPLPO或B2M作为内参基因。

Reference genes are often used to adjust gene expression in gene expression analysis.However,the expression of reference genes is not stable under different conditions.The purpose of this experiment is to select the stable expression of the reference genes in different tissues of Guangling Large sheep.The skin,perirenal fat,liver and Longissimus dorsal muscle of 6 months old Guangling Large sheep were selected as test samples.The mRNA abundance of GAPDH、18S rRNA、B2M、ACTβ、RPLPO and YWHAZ were measured by real-time quantitative PCR.The stability of the reference gene was analyzed by geNorm,NormFinder and BestKeeper software.The results showed that in the Longissimus dorsal muscle and perirenal fat of Guangling Large sheep,the expression of GAPDH was the most stable in three softwares;in the liver,the expression of RPLPO was the most stable in three softwares;in the skin,the expression of 18S rRNA was the most stable in both geNorm and NormFinder,while the expression of GAPDH was the most stable in both geNorm and BestKeeper,so was selected 18S rRNA or GAPDH were used as the reference genes;when comparing the most stable genes expressed in four tissues,the software of geNorm and NormFinder showed that the expression of RPLPO was the most stable,while the best keeper analysis showed that the stability of B2M gene was the best,so RPLPO or B2M was selected as the reference gene.the above results select the stable expression of reference genes in Guangling Large sheep skin,perirenal fat,liver,Longissimus dorsal muscle and four tissues,providing reference for the follow-up quantitative expression analysis of Guangling Large sheep genes.