Smits Katrien, Goossens Karen, Van Soom Ann, Govaere Jan, Hoogewijs Maarten, Vanhaesebrouck Emilie, Galli Cesare, Colleoni Silvia, Vandesompele Jo, Peelman Luc
Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium.
BMC Res Notes. 2009 Dec 11;2:246. doi: 10.1186/1756-0500-2-246.
Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos.
The expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32, SDHA, TUBA4A, UBC) was determined in 3 populations of equine blastocysts (fresh in vivo, fresh and frozen-thawed in vitro embryos). Application of geNorm indicated UBC, GAPDH, ACTB and HPRT1 as the most stable genes in the in vivo embryos and UBC, RPL32, GAPDH and ACTB in both in vitro populations. When in vivo and in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH were found to be the most stable. SDHA and H2A/I appeared to be highly regulated.
Based on these results, the geometric mean of UBC, ACTB, RPL32 and GAPDH is to be recommended for accurate normalization of quantitative real-time PCR data in equine in vivo and in vitro produced blastocysts.
逆转录定量实时聚合酶链反应的应用非常适合揭示体内和体外生产胚胎之间的基因表达差异。最终，这可能会优化马的辅助生殖技术。然而，为了正确解释实时PCR结果，所有数据都必须进行标准化，最可靠的方法是计算最稳定的参考基因的几何平均值。在本研究中，为马的体内胚胎以及新鲜和冻融后的体外胚胎鉴定了一组可靠的参考基因。
在3组马囊胚（体内新鲜胚胎、新鲜和冻融后的体外胚胎）中测定了8个候选参考基因（ACTB、GAPDH、H2A/I、HPRT1、RPL32、SDHA、TUBA4A、UBC）的表达稳定性。geNorm分析表明，UBC、GAPDH、ACTB和HPRT1是体内胚胎中最稳定的基因，而UBC、RPL32、GAPDH和ACTB在两个体外胚胎群体中最稳定。当将体内和体外胚胎合并分析时，发现UBC、ACTB、RPL32和GAPDH是最稳定的。SDHA和H2A/I似乎受到高度调控。
基于这些结果，推荐使用UBC、ACTB、RPL32和GAPDH的几何平均值，以准确标准化马体内和体外生产囊胚的定量实时PCR数据。
