大蒲莲猪不同发育阶段组织RT-qPCR分析中适宜内参基因筛选

Selection of Appropriate Reference Genes for RT-qPCR Analysis in Different Tissue with Different Development Periods in Dapulian Pigs

利用适宜数量且表达稳定的内参基因对目的基因的表达量进行标准化是获得准确、可靠RT-qPCR数据的重要条件。以不同日龄大蒲莲猪8种组织(心脏、肝脏、脾脏、肺脏、肾脏、肠组织、肌肉、血液)为试验材料,使用ge Norm、Norm Find软件对8个常用候选内参基因(ACTB、B2M、GAPDH、RPL4、SDHA、TBP、YWHAZ、PPIA)的表达稳定性进行分析,筛选适宜内参基因及其组合。ge Norm、Norm Find软件联合分析结果表明,不同日龄大蒲莲猪心脏中表达最稳定的内参基因为TBP、PPIA和YWHAZ,肝脏、肾脏、肠组织和肌肉中最稳定的内参基因均为TBP和PPIA,脾脏中表达最稳定内参基因为TBP和RPL4,肺脏中表达最稳定内参基因为TBP、RPL4、PPIA和ACTB,血液中表达最稳定内参基因为ACTB、YWHAZ和GAPDH。由于大蒲莲猪8种体组织中最适宜内参基因组合和数量不同,因此内参基因筛选是进行RT-qPCR数据分析的必要条件。结果可为研究大蒲莲猪目的基因表达时适宜内参基因的选择提供帮助。

To obtain the accurate RT-qPCR results,the expression levels of genes of interest should be normalized with appropriate and optimal numbers of reference genes.In this study,using two excel-based programs,geNorm and NormFind,we assessed the expression stability of eight well-known candidate reference genes( ACTB,B2M,GAPDH,RPL4,SDHA,TBP,YWHAZ and PPIA )in eight body tissues(heart,liver,spleen,lung,kidney,intestines,muscle and peripheral blood)with different development periods of Dapulian pigs.Combination analysis of two programs showed that the stable and appropriate reference genes were TBP , PPIA and YWHAZ in heart;TBP and PPIA in liver,kidney,intestines and muscle;TBP and RPL4 in spleen;TBP , RPL4 , PPIA and ACTB in lung;ACTB , YWHAZ and GAPDH in peripheral blood.The difference of suitable number and composition reference genes in eight body tissues of Dapulian indicated that the selection studies of optimal reference genes were essential in RT-qPCR analysis.The results provided reliable reference genes for expression studying of various genes related to diseases,meat quality,litter size and reproduction in Dapulian pigs.