干扰MSTN的牛骨骼肌卫星细胞实时荧光定量PCR内参基因的筛选

Selection of Reference Genes for Real-Time Quantitative PCR in Bovine Skeletal Muscle Satellite Cells Interfering with MSTN

为了筛选在牛骨骼肌卫星细胞(MSCs)分化前后稳定表达以及不受MSTN基因表达影响的内参基因,试验以野生组(WT)、转染干扰MSTN组(si-MSTN)和对照组(NC-MSTN)的牛MSCs作为样品,选取HMBS、B2M、GAPDH、TUBB、SDHA、18S rRNA、ACTB、RPL4、PPIA、HPRT1和YWHAZ作为候选内参基因,采用实时荧光定量PCR技术Ct值分析法对各候选内参基因的相对表达进行测定,首先利用3个独立评价软件geNorm、NormFinder和BestKeeper分别对各候选内参基因在野生组牛MSCs增殖期(GM)和分化第3天(DM3)细胞中的表达稳定性进行评价,筛选出前5个表达相对稳定的候选内参基因;然后以此为基础,利用相同的方法分析MSTN干扰组和对照组增殖期和分化第3天的细胞中上述5个内参基因的表达水平和稳定性。geNorm、NormFinder分析结果均显示,HMBS、B2M、TUBB、GAPDH和ACTB在牛MSCs分化前后表达较稳定,BestKeeper分析显示ACTB和TUBB相关系数(r)排序靠前,GAPDH、HMBS和B2M排序不是很高,但是GAPDH的SD值最小,因此选择这5个候选内参基因做后续试验;在牛MSCs MSTN干扰组和对照组增殖期和分化第3天,geNorm、NormFinder软件分析结果显示,上述5个候选内参基因中GAPDH、TUBB和B2M表达最稳定,BestKeeper分析显示TUBB相关系数排名不是最高,但其SD值最小,综合以上分析,选择TUBB作为牛MSCs干扰组和对照组增殖期和分化第3天最适内参基因。研究结果可为以后进行牛MSCs在不同生长时期以及MSTN表达被调控后基因的表达分析提供参考。

In order to screen the reference genes that suitable for bovine skeletal muscle satellite cells(MSCs)differentiation process and not affected by MSTN gene expression,bovine MSCs from wild group(WT),MSTN interfered group(si-MSTN)and control group(NC-MSTN)were used as sample.HMBS,B2M,GAPDH,TUBB,SDHA,18S rRNA,ACTB,RPL4,PPIA,HPRT1 and YWHAZ were selected as candidate internal reference genes.The relative expression of candidate internal reference genes was determined by Real-time quantitative PCR and Ct value analysis.Firstly,the expression stability of each candidate internal reference gene in wild group(WT)bovine MSCs of proliferative(GM)and the differentiated third day after differention(DM3)cells was evaluated by three independent evaluation softwares:geNorm,NormFinder and BestKeeper,and the first five candidate reference genes with the most stable expression were screened out.Furthermore,the expression level and stability of these five internal reference genes in si-MSTN group and NC-MSTN group of GM and DM3 were analyzed by similar methods.The analysis of geNorm and NormFinder showed that the expression of HMBS,B2M,TUBB,GAPDH and ACTB genes were more stable before and after differentiation of bovine MSCs.The BestKeeper showed that the correlation coefficient(R)of ACTB and TUBB were ranked first,GAPDH,HMBS and B2M were not very high,but GAPDH had the lowest SD value,so these five candidate genes were selected for subsequent experiments.In siRNA-MSTN group and NC-MSTN group of GM and DM3,the results of geNorm and NormFinder showed that the expressions of GAPDH,TUBB and B2M in the five candidate reference genes were more stable than other.BestKeeper showed that the correlation coefficient of TUBB was not the highest,but its SD value was the lowest.Based on the above analysis,TUBB was selected as the most suitable internal reference gene in GM and DM3 of bovine MSCs siRNA-MSTN and NC-MSTN groups.The result of this study would provide reference for future research on gene expression analysis of bovine MSCs in different growth process and after MSTN expression was regulated.