实时荧光定量PCR所用仔猪组织内参基因的筛选

Screening of the housekeeping gene of real-time quantitative PCR in piglet tissues

为了保证在应用实时荧光定量PCR方法检测仔猪目的基因时,检测结果可以科学地用于多组织间的纵向比较,减少因内参基因在多组织间变化而带来的误差,筛选出能够在多组织间稳定表达的内参基因,对于目标基因的准确校正具有重要意义。本试验以4周龄仔猪不同组织(心、肝、脾、肺、肾、胰、气管、骨骼肌、肺门淋巴结、大肠、小肠、大脑、小脑)为试验样本,利用qPCR方法分别检测3-磷酸甘油醛脱氢酶(GAPDH)、泛素辍合酶(UBC)、核糖体蛋白L32(RPL32)、核糖体蛋白L3(RPL3)、乳酸脱氢酶A(LDHA)、次黄嘌呤鸟嘌呤磷酸核糖转移酶1(HPRT1)、羟甲基胆素合成酶(HSBM)等7个基因在各组织内的m RNA表达稳定情况。经LinReg PCR和geNorm软件统计分析,结果表明,上述看家基因在所选的13种组织中表达稳定性依次为RPL3>RPL32>UBC>HSBM>HPRT1>GAPDH>LDHA。综上所述,RPL3基因在13种组织中的相对稳定性最高。

It is important to screen out a housekeeping gene that can express stably in different tissues for accurate correction of target genes expression and avoiding the error causing by the variety of the housekeeping gene expression in different tissues,when detecting gene expression in piglet via real-time quantitative PCR.In present study,different tissues(heart,liver,spleen,lung,kidney,pancreas,trachea,skeletal muscle,hilar lymph node,large intestine,small intestine,brain,cerebellum) from four weeks old piglets were used as the experimental samples to detect the stability of m RNA expression of seven genes, glyceraldehyde-3-phosphate dehydrogenase(GAPDH),ubiquitin conjugating enzyme(UBC),ribosomal protein L32(RPL32),ribosomal protein L3(RPL3),lactate dehydrogenase A(LDHA),hypoxanthine phosphoribosyltransferase1(HPRT1),hydroxymethylbilane synthase(HMBS).Statistical analysis,which was carried out by software Lin Reg PCR and ge Norm,showed that the order of the stability of 7 housekeeping genes was RPL3>RPL32>UBC>HSBM> HPRT1>GAPDH>LDHA among the 13 kinds of tissues.In conclusion,RPL 3 gene has the highest relative stability.