二倍体和四倍体泥鳅qRT-PCR分析中内参基因优选

Screening of optimal internal reference genes for qRT-PCR in diploid and tetraploid loach,Misgurnus anguillicaudatus

为筛选在泥鳅中稳定表达的内参基因,以确保泥鳅基因表达分析结果的可靠性,选取ACTB、TUBA、EF1A、GAPDH、TUBB和18SrRNA6个常用的内参基因作为候选内参基因,分别用BestKeeper、geNorm、NormFinder、ΔCt和RefFinfer软件分析这6个基因在二倍体和四倍体泥鳅胚胎发育阶段、成鱼组织间以及倍性间表达的稳定性。结果显示,在胚胎发育各阶段,TUBB和TUBA的表达最稳定,可作为胚胎发育阶段qRT-PCR分析的内参基因;其中,TUBB在泥鳅倍性间的表达差异不显著,因此,可作为泥鳅胚胎发育中跨倍性qRT-PCR研究的内参基因。在成鱼各组织中,ACTB和18SrRNA的表达最稳定,可用作组织间qRT-PCR分析的内参基因;其中,18SrRNA在倍性间的组织表达差异不显著,因此,可作为泥鳅组织间跨倍性qRT-PCR的内参基因。

To identify the optimal internal reference genes for qRT-PCR analysis in loach ( Misgurnus anguillicaudatus ),six commonly used reference genes,including ACTB,TUBA,EF1A,GAPDH,TUBB and 18S rRNA were selected as candidates,and their expression stability during different developmental stages,across-tissues and between-ploidy were evaluated by using BestKeeper,geNorm,NormFinder,ΔCt and RefFinfer software.The results showed that,during the process of embryonic development,the expression levels of TUBB and TUBA were more stable than the other genes,suggesting that they can be used as reference genes.Besides,the expression level of TUBB showed no significant difference between diploids and tetraploids ( P >0.05),suggesting it is the most suitable internal reference genes for qRT-PCR analysis during embryonic development between-ploidy.Among different tissues of diploids and tetraploids,the expression levels of ACTB and 18S rRNA were more stable than the other genes,thus they can be used as internal control genes.Of which,18S rRNA was considered as the most suitable internal control genes,because the expression level of it exhibited no significant difference in different tissues between diploids and tetraploids ( P >0.05).