小鼠睾丸荧光定量PCR分析的内参基因筛选

Screening of reference genes in mice testis for quantitative RT-PCR analysis

旨在筛选出小鼠睾丸组织中最优内参基因,为研究小鼠睾丸发育过程中的目的基因表达提供基础依据.以小鼠作为研究对象,采用Trizol法提取4个不同发育时期(1周龄、3周龄、6周龄、9周龄)睾丸组织总RNA并反转录获得其cDNA.且根据已有报道,选出小鼠常见相对稳定的7种内参基因β-肌动蛋白(β-actin)、肽基异构酶A(PPIA)、酸性核糖体磷酸蛋白(ARBP)、18S核糖体RNA(18S rRNA)、琥珀酸脱氢酶复合黄蛋白亚基A(SDHA)、甘油醛-3-磷酸脱氢酶(GAPDH)和次黄嘌呤磷酸核糖转移酶1(HPRT1)的GenBank登陆序列设计所需荧光定量引物,结合实时荧光定量PCR(RT-qPCR)技术分别验证候选基因引物特异性并构建以睾丸组织为模板各基因的标准曲线,并通过GeNorm程序对各基因稳定性进行分析,筛选出小鼠睾丸中最适内参基因.结果表明,各个时期睾丸组织RNA电泳条带没有出现弥散和拖带的现象;所设计的候选基因引物条带单一没有杂带且RT-qPCR溶解曲线均呈现单一信号峰,并具有良好的线性关系;候选基因稳定性值(M值)进行排序:PPIA=β-actin>ARBP>HPRT1>SDHA>18S rRNA>GAPDH,获得小鼠睾丸中稳定性最好的2个内参基因(PPIA和β-actin).综上表明,本研究成功筛选出小鼠睾丸发育过程中稳定表达的内参基因.

This research was aimed at finding the optimal internal reference genes in mouse testis,which may serve as a primary study for studying the expression of target genes during the maturation of mouse testis.Mice were chosen as experiment animals.The Trizol reagent was used to extract total RNA from testis at 4 different stages of maturation(1 week,3 week,6 week,and 9 week),and then reverse polymerase was used to synthesize the cDNA.According to previous studies,seven commonly used internal reference genes were selected(β-actin,GAPDH,PPIA,ARBP,18S rRNA,SDHAand HPRT1).The primers were designed based on the genomes in GenBank.Moreover,the specificity of each primer was tested by the real-time quantitative PCR(RT-qPCR),and then the standard curve was constructed.The GeNorm software was used to analyze the stability of gene expression.Furthermore,the most suitable internal reference gene in mouse testis was selected.The result showed that the electrophoresis bands of RNA,extracted from samples in different stages of testis maturing,all performed well.The amplification results of reference genes presented by single clear electrophoresis bands showed good specificity.The M values of candidate reference genes were analyzed and ranked by GeNormas following:PPIA=β-actin>ARBP>HPRT1>SDHA>18S rRNA>GAPDH.This study demonstrated that the genes with best expression stability were PPIA andβ-actin.In conclusion,genes were successfully identified as the most suitable reference genes during the maturation of testis in mice.