天府肉鹅母系不同阶段颗粒细胞内参基因的选择

Identification for internal reference genes in different periods of granulosa cells of Tianfu meat geese

为筛选出鹅卵泡不同阶段颗粒细胞稳定表达的内参基因,以产蛋期天府肉鹅母系卵泡颗粒层细胞为材料,利用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, RT- qPCR)技术分别对GAPDH、ACTB、TUB、UBC、HMBS、SDH、18S、28S、TBP和HPRT1等10个候选内参基因的相对表达量进行测定,采用ΔCT法、qbase+、NormFinder、BestKeeper等4种评价方法对9个不同阶段卵泡颗粒细胞的表达稳定性进行评定。RT- qPCR熔解曲线和PCR扩增显示,10个候选内参基因的引物特异性良好;通过构建标准曲线,表明各内参基因在系列稀释的浓度梯度内具有良好的线性关系;综合4种方法的评价结果,发现在不同阶段颗粒细胞中,最稳定的3个内参基因依次为SDH、HMBS和18S,稳定性最差的3个内参基因依次为UBC、GAPDH和TUB。在不同阶段颗粒细胞中最稳定的内参基因为SDH和HMBS,以最稳定的2个内参基因的几何平均数作为标准化校正因子可得到更加准确的结果。

In order to screen out the most stable reference genes in different periods of granulosa cells in goose, we selected 10 candidate reference genes (GAPDH, ACTB, TUB, UBC, HMBS, SDH, 18S, 28S, TBP, HPRT1) to determine the relative expression levels by the real-time fluorescence quantitative polymerase chain reaction (RTqPCR). The expression stabilities of 10 candidate reference genes in nine different stages of granulosa cells were systematically analyzed by delta-CT, qbase+, NormFinder and BestKeeper, respectively. The results of RT-qPCR melting curve and PCR amplification showed that the 10 candidate reference genes were specifically amplified. By constructing a standard curve, between Cq value and the logarithm of relative copy number exhibited a good linear relationship in the serial dilution concentration gradient. Based on the evaluation results of four different algorithms, SDH, HMBS and 18S were found to be three of the most stable reference genes, but UBC, GAPDH and TUB were three of the least stable reference genes in the different periods of granulosa cells. Therefore, the most stable internal reference genes were SDH and HMBS in granulosa cells at different developmental stages,and it could get more accurate normalization of RT-qPCR data by geometric averaging of the most stable reference genes.