Brito A B, Lima J S, Brito D C, Santana L N, Costa N N, Miranda M S, Ohashi O M, Santos R R, Domingues S F S
Laboratory of Wild Animal Biology and Medicine, Universidade Federal do Pará, Brazil.
Zygote. 2013 May;21(2):167-71. doi: 10.1017/S0967199411000748. Epub 2012 Apr 5.
There is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.
在研究中，尚无传统报道过接触冷冻保护剂或单纯的缺氧和低温对卵巢组织基因表达的影响，并且仅有一项关于参考基因或靶基因定量以及常氧与缺氧、低温和毒性条件比较的研究。我们在本研究中的目的是调查卷尾猴（僧帽猴）卵巢组织中三个参考基因的稳定性。为此，使用了新鲜的和接触过冷冻保护剂的卵巢活检组织。对新鲜的和接触过冷冻保护剂的卵巢组织都进行了总RNA提取和cDNA合成。通过实时聚合酶链反应（PCR）扩增cDNA，并使用GeNorm、BestKeeper和NormFinder软件评估甘油醛-3-磷酸脱氢酶（GAPDH）、次黄嘌呤磷酸核糖基转移酶1（HPRT1）和TATA结合蛋白（TBP）的稳定性。结果表明，在卷尾猴的卵巢组织中，HPRT1和TBP是最合适的参考基因，因此可作为未来研究中数据标准化的参数。相比之下，GAPDH在测试的参考基因中是最不稳定的基因。总之，HPRT1和TBP是卷尾猴新鲜的和接触过冷冻保护剂的卵巢组织中最稳定的参考基因。
