小鼠脂肪沉积过程中定量PCR内参基因的筛选

Screening of reference genes for quantitative PCR in adipose deposition of mice

通过筛选小鼠不同类型脂肪组织及脂肪细胞诱导分化过程中的最佳内参基因,为研究小鼠脂肪沉积过程中基因相对定量表达水平提供依据。首先利用荧光定量PCR技术检测9个候选内参基因在脂肪组织及脂肪细胞诱导分化过程中的表达并构建标准曲线,然后通过RefFinder程序评估内参基因的稳定性并确定最稳定的内参基因,最后以筛选的稳定内参基因进行不同类型脂肪组织组蛋白去甲基化酶KDM2A的相对定量表达,并与KDM2A的绝对定量结果比较,评价筛选内参基因的定量效果。结果显示,RPL13a和18S rRNA分别是小鼠脂肪组织和脂肪细胞中表达最稳定的内参基因,而ACTB不适合作为小鼠脂肪沉积过程中的内参基因。采用RPL13a作为内参基因的KDM2A相对定量结果显示,KDM2A在附睾脂肪组织(eWAT)具有较高的表达量,棕色脂肪组织(BAT)次之,与KDM2A在不同类型脂肪组织的绝对定量结果一致。因此,RPL13a和18S rRNA可作为小鼠脂肪组织及脂肪细胞基因相对定量的最适内参基因。

The aim of this study was to screen the best reference genes among different depots and during adipocyte adipogenic differentiation in mice,so as to provide evidences for relative quantitative expression of genes using qRT-PCR in mouse fat deposition.Firstly,the expression of 9 candidate reference genes in adipose tissue and adipocyte induced differentiation was detected by qRT-PCR and the standard curve was constructed.Then RefFinder was used to evaluate the stability of detected reference genes,and finally,the most stable reference gene was determined,which is verified by absolute quantitative of KDM2 A among BAT,iWAT and eWAT.The results showed that RPL13 a and 18 S rRNA were the most stable reference genes in mouse adipose tissue and adipocyte,respectively.ACTB was not suitable as a reference gene in mouse fat deposition.Additionally,the relative quantitative expression of KDM2 A in different adipose tissues using RPL13 a showed that KDM2 A is highest in eWAT,which is consistent with the absolute quantitative expression pattern of KDM2 A in different adiposes.Therefore,RPL13 a and 18 S rRNA were the suitable reference genes for relative gene expression measurement of adipose tissues and adipocytes adipogenesis,respectively.