民猪外周血单核细胞实时荧光定量PCR内参基因的筛选

摘要

Min pig is a local pig breed in Northeast China.It has been well-adapted to the local cold weather,but few genes related to its environmental adaptation have been studied.For studies about environmental adaptation of Min pig on molecular level,it is important to have proper refer ence genes for quantification of gene expression by quantitative Real-time PCR.In this study,12 reference genes (B2M,ACTB,RPL11,RPL4,YWHAZ,GAPDH,HPRT1,SDHA,HMBS,IDH3B,TUBB2B and TBP1) were evaluated for their potential as the reference gene in Min pig peripheral blood mononuclear cells under different temperatures.Blood samples were collected from 3 Min pigs which were under-25,5,10 and 30 C,respectively.Mononuclear cells were separated using density gradient centrifugation.Statistical algorithms including geNorm,Normfinder and BestKeeper were employed to assess the stabilities of these genes.Analysis of geNorm and Normfinder revealed that all these 12 genes were highly stable.However,ACTB,GAPDH,SDHA,HPRT1,TBP1 and YWHAZ genes (SD＜1) were found to be more stable than other six genes (SD＞1),of which TBP1 was the most stable one using BestKeeper program.To summarize,ACTB,GAPDH,SDHA,HPRT1,TBP1 and YWHAZ genes were suitable to be the reference genes,with TBP1 was the best one.%民猪是能够适应东北地区寒冷环境的地方猪种,但目前对其环境适应性相关基因的研究较少,也没有确定适合此研究所需的实时荧光定量PCR的内参基因.因此,本试验通过研究常用的12个内参基因(B2M、ACTB、RPL11、RPL4、YWHAZ、GAPDH、HPRT1、SDHA、HMBS、IDH3B、TUBB2B和TBP1)在不同环境温度下民猪外周血单核细胞中的表达稳定性,旨在确定合适的内参基因进行相关研究.分别在-25、5、10和30℃采集3头民猪耳静脉血分离出单核细胞,利用geNorm、NormFinder、BestKeeper 3种软件分析12个候选内参基因的Ct值,筛选出表达稳定的基因作为内参基因.经geNorm和NormFinder计算获得各候选基因的M值发现,12个候选基因的表达均相对稳定,而BestKeeper的分析则显示ACTB、GAPDH、SDHA、HPR T1、TBP1、YWHAZ基因的SD值均＜1,可用作本研究条件下的内参基因,而另外6个基因SD值则＞1,不符合作为内参基因的标准.综合3种分析的结果,在本研究条件下,TBP1基因的稳定性最高,ACTB、GAPDH、SDHA、HPRT1和YWHAZ基因也符合作为内参基因的标准,都可研究在不同环境温度下民猪外周血单核细胞中基因表达时作为内参基因.