猪卵泡发育过程中颗粒细胞内参基因表达稳定性分析

Stability analysis of reference gene expression in granulosa cells during porcine follicular development

[目的]本研究旨在分析猪卵泡发育不同阶段颗粒细胞中内参基因的表达稳定性,筛选有效内参基因用于鉴定猪卵泡发育关键调控基因及其表达模式。[方法]基于形态学观察与类固醇激素检测分离猪腔前、有腔、成熟和闭锁卵泡,并收集相应颗粒细胞;利用RT-qPCR检测甘油醛3-磷酸脱氢酶(GAPDH)、β-肌动蛋白(ACTB)、ATP合酶F1(ATP5F1)、β2-微球蛋白(B2M)、真核翻译伸长因子1α(EEF1A1)、次黄嘌呤磷酸核糖转移酶1(HPRT1)、核糖体蛋白S3(RPS3)、微管蛋白α-1b(TUBA1B)和泛素C(UBC)等9个候选内参基因在猪卵泡不同发育阶段颗粒细胞中的表达水平;综合ΔCt、geNorm、NormFinder、BestKeeper和RefFinder等算法分析候选内参基因在猪卵泡发育过程中的表达稳定性。[结果]候选内参基因在猪卵巢颗粒细胞中的表达水平存在较大差异,其中EEF1A1和TUBA1B的表达水平较高,而HPRT1和UBC的表达水平较低;不同算法得到的稳定性存在差异,用ΔCt、NormFinder和BestKeeper分析发现RPS3和ATP5F1的稳定性较高,用geNorm分析发现GAPDH和HPRT1的稳定性较高,最终利用RefFinder进行综合权重分析得出候选内参基因的稳定性从高到低依次为RPS3、ATP5F1、HPRT1、EEF1A1、GAPDH、ACTB、TUBA1B、B2M、UBC。进一步以不同表达稳定性内参基因为参照对母猪繁殖力候选基因FSHR和NORFA在卵泡发育过程中的表达模式进行检测,结果表明内参基因稳定性对准确鉴定目的基因表达水平和变化模式至关重要。[结论]RPS3和ATP5F1在猪卵泡发育过程中具有较高的表达稳定性,可作为有效内参基因用于后续猪卵泡发育相关研究,为鉴定猪卵泡发育关键调控基因及其表达模式等相关内参基因研究提供理论依据。

[Objectives]The aim of this study was to analyze the expression stability of reference genes in porcine granulosa cells(GC)during follicular development,and to further screen effective reference genes(RG)for the identification of key regulatory genes and their expression patterns.[Methods]Porcine preantral,antral,mature and atretic follicles were isolated and classified based on morphological observation with steroid hormone detection,and corresponding GC were collected.RT-qPCR was performed to detect the expression levels of nine candidate RG,including glyceraldehyde-3-phosphate dehydrogenase(GAPDH),actin beta(ACTB),tubulin alpha-1b(TUBA1B),ATP synthase f1(ATP5F1),beta-2-microglobulin(B2M),ubiquitin C(UBC),hypoxanthine phosphoribosyltransferase 1(HPRT1),ribosomal protein S3(RPS3),and eukaryotic translation elongation factor 1 alpha(EEF1A1);ΔCt,geNorm,NormFinder,BestKeeper and RefFinder algorithms were used to analyze the expression stability of candidate RG in porcine GC during follicle development.[Results]The expression levels of candidate RG in porcine GC were significantly different,among which EEF1A1 and TUBA1B had higher expression levels,while HPRT1 and UBC had lower expression levels.ΔCt,NormFinder and BestKeeper analysis showed that RPS3 and ATP5F1 were the most stable RG,while GAPDH and HPRT1 were found to be more stable by geNorm analysis.Finally,the comprehensive weighted analysis by RefFinder showed that the stability of the candidate RG was ranked as follows:RPS3,ATP5F1,HPRT1,EEF1A1,GAPDH,ACTB,TUBA1B,B2M and UBC.Furthermore,the expression patterns of fertility-associated candidate genes(FSHR and NORFA)during follicular development were analyzed with different RG as controls,and results showed that the stability of RG was important for accurate identification of the expression and patterns of target genes.[Conclusions]RPS3 and ATP5F1 with higher expression stability could be used as effective RG for subsequent studies on sow follicular development,which also provide a theoretical basis for ideal RG selection to identify the key regulatory genes and their expression patterns during sow follicular development.