实时定量PCR研究中需要选择表达稳定性高的内参基因才能准确地校正目标基因的表达量。以仔猪不同组织为研究对象,应用实时荧光定量PCR技术,检测了beta-肌动蛋白(ACTB)、beta-2-微球蛋白(B2M)、3-磷酸甘油醛脱氢酶(GAPDH)、羟甲基胆素合成酶(HMBS)、次黄嘌呤鸟嘌呤磷酸核糖转移酶1(HPRT1)、核糖体蛋白L4(RPL4)、琥珀酸脱氢酶(亚基A)(SDHA)、TATA盒结合蛋白1(TBP1)和酪氨酸3-单氧化酶/色氨酸5-单氧化酶激活蛋白zeta多肽(YWHAZ)共9个看家基因mRNA水平的表达情况。经geNorm程序统计学分析处理,结果表明,9个看家基因的表达稳定性为HMBS和HPRT1〉RPL4〉TBP1〉B2M〉YWHAZ〉SDHA〉GAPDH〉ACTB,确定了HMBS基因在仔猪不同组织中用于校正目标基因的表达量为最佳选择,而RPL4基因则可以作为高转录本的内参基因。
Selection of the best stable reference genes is of crucial importance for the accurate normalization of expression in real-time quantitative PCR.Taking the piglet tissues as example,the expressions of nine housekeeping geens beta-actin (ACTB),beta-2-microglobulin (B2M),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),hydroxymethylbilane synthase(HMBS),hypoxanthine phosphoribosyltransferase 1(HPRT1),ribosomal protein L4 (RPL4),succinate dehydrogenase complex,subunit A (SDHA),TATA box binding protein1 (TBP1) and Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein and zeta polypeptide (YWHAZ) were detected using real-time quantitative PCR.And their expression stability were observed by analysis of geNorm program.Results showed HPRT1 and HMBS〉 RPL4 〉TBP1〉 B2M〉 YWHAZ〉 SDHA〉 GAPDH〉 ACTB.As the best choice,HMBS can be used as normalizing expression of target gene in different piglet tissues,while RPL4 is good reference gene for high abundant transcripts.