小鼠玻璃化冷冻卵巢实时定量PCR分析中内参基因的选择
Reference gene selection for real-time quantitative PCR analysis on ovarian cryopreservation by vitrification in mice.
作者信息
Yuanyuan Shan, Qin Su, Rongrong Xu, Yujing Gao, Chengbin Pei, Jianjun Ma, Yanzhou Yang, Xiuying Pei
机构信息
Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Key Laboratory of Reproduction and Genetics in Ningxia, Department of Biochemistry and Molecular Biology, Ningxia Medical University, Yinchuan, Ningxia, 75004, People's Republic of China.
出版信息
J Assist Reprod Genet. 2015 Aug;32(8):1277-84. doi: 10.1007/s10815-015-0503-5. Epub 2015 Jun 27.
PURPOSE
To ensure the correct interpretation of the results of quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) from ovarian tissue cryopreserved by vitrification, it is critical to normalize expression levels to a reference gene with stable messenger RNA (mRNA) expression in the vitrified/warmed ovarian tissue. The aim of this work was to identify suitable reference genes for qRT-PCR analysis during ovarian cryopreservation by vitrification.
METHODS
GeNorm, NormFinder, comparative Delta-CT, and BestKeeper were used to analyze the expression and stability of the 14 reference genes GAPDH, ABL1, ACTB, CDKN1A, GPER, GUSB, HPRT1, HSP90AB1, IPO8, PPIA, RPL4, RPL30, TBP, and UPAR.
RESULTS
Our results indicated that ACTB and RPL4 were relatively stable reference genes in vitrified/warmed ovaries.
目的
为确保对经玻璃化冷冻保存的卵巢组织进行定量实时逆转录-聚合酶链反应（qRT-PCR）结果的正确解读，至关重要的是将表达水平标准化至在玻璃化/复温卵巢组织中具有稳定信使核糖核酸（mRNA）表达的参考基因。本研究的目的是鉴定在卵巢玻璃化冷冻保存期间用于qRT-PCR分析的合适参考基因。
方法
使用GeNorm、NormFinder、比较Δ-CT和BestKeeper分析14个参考基因甘油醛-3-磷酸脱氢酶（GAPDH）、阿伯尔逊鼠白血病病毒癌基因同源物1（ABL1）、β-肌动蛋白（ACTB）、周期蛋白依赖性激酶抑制剂1A（CDKN1A）、雌激素相关受体（GPER）、β-葡萄糖醛酸酶（GUSB）、次黄嘌呤磷酸核糖转移酶1（HPRT1）、热休克蛋白90α家族成员1（HSP90AB1）、IPO8、肽基脯氨酰异构酶A（PPIA）、核糖体蛋白L4（RPL4）、核糖体蛋白L30（RPL30）、TATA盒结合蛋白（TBP）和尿激酶型纤溶酶原激活物受体（UPAR）的表达和稳定性。
结果
我们的结果表明，ACTB和RPL4是玻璃化/复温卵巢中相对稳定的参考基因。
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