南京农业大学学报2024，Vol.47Issue(4)：710-720,11.DOI:10.7685/jnau.202306019
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
摘要
Abstract
[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.关键词
猪/卵泡发育/卵巢颗粒细胞/RT-qPCR/内参基因/表达稳定性分析Key words
porcine/follicle development/granulosa cells/RT-qPCR/reference genes/expression stability analysis分类
农业科技引用本文复制引用
霍泱安,李小雪,孙郴,李齐发,杜星..猪卵泡发育过程中颗粒细胞内参基因表达稳定性分析[J].南京农业大学学报,2024,47(4):710-720,11.基金项目
国家自然科学基金项目(32372839,31902130) （32372839,31902130）
江苏省自然科学基金项目(BK20231475) （BK20231475）
