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	17α,20β双羟孕酮诱导鲤卵母细胞最终成熟相关基因表达内参基因的筛选; Reference gene validation for quantification of gene expression during final oocyte maturation induced by 17α, 20β-dihydroxy-4-pregnen-3-one in common carp(Cyprinus carpio)
	史艳艳 ; 卢洁 ; 王艺磊 ; 王淑红
	2014-09-15
关键词	鲤 17α 20β双羟孕酮 基因表达 内参基因 卵母细胞最终成熟 实时荧光定量PCR Cyprinus carpio 17α 20β-dihydroxy-4-pregnen-3-one gene expression reference gene final oocyte maturation quantitative real-time PCR
英文摘要	本研究检测了18S rrnA(18S)、28S rrnA(28S)、组织蛋白酶z(CATHEPSIn z,CTSz)、延伸因子1-α(ElOngATIOn fACTOr 1-α,Ef-1α)、3-磷酸甘油脱氢酶(glyCErAldEHydE-3-PHOSPHATE dEHydrOgEnASE,gAPdH)、β肌动蛋白(β-ACTIn)6个候选内参基因在17α,20β双羟孕酮(dHP)诱导鲤(CyPrInuS CArPIO)卵母细胞最终成熟过程的表达情况,并应用不同的内参分析软件对数据进行了分析。bESTkEEPEr软件分析表明Ef-1α的变异系数和标准差是6个候选内参基因中最低的,且bESTkEEPEr指数在6个候选内参基因中最高,说明其表达稳定性最高,适合做为内参基因;gEnOrM软件分析认为需要同时使用Ef-1α和CTSz两个内参基因来校正目标基因的表达;nOrMfIndEr软件分析同样表明Ef-1α是6个候选内参基因中表达最稳定的;最后通过rEffIndEr软件综合比较分析,确定Ef-1α相对于其他5个候选内参基因,在17α,20β双羟孕酮诱导卵母细胞最终成熟阶段表达最为稳定,可作为内参校正17α,20β双羟孕酮诱导鲤卵母细胞最终成熟阶段各基因的表达情况。; Final oocyte maturation is a key step for successful spawning and fertilization.Like most other vertebrates, teleosts have full-grown postvitellogenic oocytes in the ovary that are physiologically arrested at the G2/M border in the first meiotic prophase and cannot be fertilized.Shortly before ovulation, the oocytes must become fully mature, which involves breakdown of the germinal vesicle(GVBD), chromosome condensation, assembly of the meiotic spindle, and exclusion of the first polar body.Meiosis is again arrested at the second metaphase.The time period from the resumption of meiosis to the second meiotic metaphase has been referred to as final oocyte maturation.Knowledge of the molecular events and the role of various factors involved in final oocyte maturation is a focus of research in the field of reproductive biology.Quantitative real-time PCR(qPCR) is often used to quantify transcript abundance in such studies; however the technique is subject to considerable experimental error and variation.A stably expressed housekeeping gene is typically used as a reference gene to normalize this variation.However an ideal universal reference gene that is stable under all experimental circumstances has not been described.Researchers should validate their reference genes before performing qPCR analysis.We documented the expression of six candidate reference genes: 18 S rRNA(18S), 28 S rRNA(28S), cathepsin Z(CTSZ), elongation factor 1-α(EF-1α), glyceraldehyde-3-phosphate dehydrogenase(gapdh), and β-actin during 17α, 20β-dihydroxy-4-pregnen-3-one(DHP) induced final oocyte maturation in common carp(Cyprinus carpio).We used a range of software to evaluate the expression stability of these reference genes.Analysis with Bestkeeper revealed that EF-1α had the lowest CV%(2.65) and STDEV(0.71) values, but had the highest Bestkeeper index value(0.956) among the six candidate reference genes, suggesting that EF-1α was the best reference gene in the present study.Analysis with geNorm revealed that EF-1α and CTSZ had the lowest M(gene expression stability) values which indicated that they were the most stable genes among the six.Additionally, the value of pairwise variation of these two genes(V2/3) was much lower than the default(0.15), suggesting that EF-1α and CTSZ should be used concurrently as a reference gene pair to normalize the expression of the target genes.Consistently, analysis with NormFinder and RefFinder demonstrated that EF-1α was the most stable gene among the six candidates.Hence, EF-1α can serve as a reference gene to normalize the expression of target genes during final oocyte maturation induced by DHP in common carp.The mRNA expression of luteinizing hormone receptor gene(LHR) during final oocyte maturation induced by DHP was normalized by 6 candidate reference genes respectively, and the relative expression was varied significantly depending on the reference gene that was used.Selection of a stably expressed reference gene is critical for all qPCR analysis to ensure accurate target gene mRNA expression information.; 集美大学创新团队基金项目(2010A001)
语种	zh_CN
内容类型	期刊论文
源URL	[http://dspace.xmu.edu.cn/handle/2288/105754]
专题	航空航天－已发表论文
推荐引用方式 GB/T 7714	史艳艳,卢洁,王艺磊,等. 17α,20β双羟孕酮诱导鲤卵母细胞最终成熟相关基因表达内参基因的筛选, Reference gene validation for quantification of gene expression during final oocyte maturation induced by 17α, 20β-dihydroxy-4-pregnen-3-one in common carp(Cyprinus carpio)[J],2014.
APA	史艳艳,卢洁,王艺磊,&王淑红.(2014).17α,20β双羟孕酮诱导鲤卵母细胞最终成熟相关基因表达内参基因的筛选..
MLA	史艳艳,et al."17α,20β双羟孕酮诱导鲤卵母细胞最终成熟相关基因表达内参基因的筛选".(2014).

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