Rating of putative housekeeping genes for quantitative gene expression analysis in cyclic and early pregnant equine endometrium
Rating of putative housekeeping genes for quantitative gene expression analysis in cyclic and early pregnant equine endometrium
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DOI：
10.1016/j.anireprosci.2011.02.019
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发表时间：
2011-05-01
影响因子：
2.2
通讯作者：
Guzeloglu, Aydin
中科院分区：
文献类型：
--
作者：
Kayis, Seyit A.;Atli, Mehmet O.;Guzeloglu, Aydin
目的是评估一组管家基因（HKGs），用于马子宫内膜基因表达的标准化。使用实时荧光定量聚合酶链反应（real - time PCR）对甘油醛 - 3 - 磷酸脱氢酶（GAPDH）、次黄嘌呤核糖基转移酶1（HPRT1）、泛素B（UBB）、微管蛋白α1（TUBA1）、核糖体蛋白L32（RPL32）、β - 2 - 微球蛋白（B2M）、18S核糖体RNA（18S）和28S核糖体RNA（28S）等管家基因进行评估，并在子宫内膜的不同生理阶段进行比较。在排卵日（d0，n = 4）、发情后期（LD，n = 4）、周期黄体溶解后（AL，n = 4）以及怀孕第14天（P14；n = 3）、第18天（P18，n = 3）和第22天（P22；n = 3）从母马获取子宫内膜活检样本。根据实验设计提出了一个基于约束最大似然法（REML）并辅以描述性统计学的模型，并通过主成分分析（PCA）进一步验证。将结果与广泛使用的软件（包括geNorm、BestKeeper和NormFinder）进行比较。结果表明，GAPDH是最稳定的管家基因，RPL32排名第二。18S和28S被发现是最不稳定的。所提出的模型、PCA、geNorm和BestKeeper在检测发情周期和早期妊娠期间马子宫内膜中最稳定和最不稳定的管家基因方面是一致的。（C）2011爱思唯尔有限公司。保留所有权利。
The aim was an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in the equine endometrium. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine ribosyl transferase 1 (HPRT1), ubiquitin B (UBB), tubulin alpha 1 (TUBA1), ribosomal protein L32 (RPL32), beta-2-microglobulin (B2M), 18S rRNA (18S), and 285 rRNA (28S) HKGs were evaluated using real-time PCR and were compared in different physiological stages of the endometrium. Endometrial biopsies were obtained from mares on day of ovulation (d0, n = 4), at late diestrus (LD, n = 4), after luteolyis (AL, n = 4) of the cycle and on days 14 (P14; n = 3), 18 (P18, n = 3) and 22 (P22; n = 3) of pregnancy. A model based on REML with support of descriptive statistics was proposed in accordance with experimental design and was further confirmed with principal component analysis (PCA). Results were compared with widely used software including geNorm, BestKeeper, and NormFinder. Results indicated that GAPDH was the most stable HKG and RPL32 was ranked as the second best. 18S and 285 were found to be the least stable. The proposed model, PCA, geNorm, and BestKeeper were in agreement in detecting the most stable and the least stable HKGs in the equine endometrium during the estrous cycle and early pregnancy. (C) 2011 Elsevier B.V. All rights reserved.