Leibniz Institute for Zoo and Wildlife Research (IZW), Evolutionary Genetics, Alfred-Kowalke-Str. 17, 10315 Berlin, Germany.
Biochem Biophys Res Commun. 2010 Feb 5;392(2):145-9. doi: 10.1016/j.bbrc.2009.12.173. Epub 2010 Jan 6.
Quantitative real-time RT-PCR (qRT-PCR) is a sensitive technique for gene expression analysis. A critical factor for creating reliable data in relative quantification is the normalization of the expression data of genes of interest. Therefore the needed normalization factor is calculated out of the expression data of co-amplified genes that are stable expressed in the certain sample material, the so-called reference genes. In this study, we demonstrate the important process of validating potential reference genes using a non-model species. As there are almost no sequences known of the Pallid Atlantic Forest Rat (Delomys sublineatus), a rodent used as indicator species in conservation studies of the endangered Brazilian rainforest, suitable primer sets are more problematic to find than in model species. Out of nine tested primer sets designed for the fully sequenced Mus musculus, five could be used for the establishment of a proper running SYBR-Green assay and validation of their constant expression. qRT-PCR results of 12 cDNAs of Delomys livers were analyzed with three different validation software programs: BestKeeper, NormFinder and geNorm. Our approach showed that out of the five (Sdha, Canx, Pgk1, Actb and Actg1) potential reference genes, the first four should be used for accurate normalization in further relative quantification analyses. Transferring data from close-by model organisms makes high sensitive real-time RT-PCR applicable even to free-ranging non-model organisms. Our approach might be suitable for other non-model organisms.
实时荧光定量 RT-PCR（qRT-PCR）是一种用于基因表达分析的敏感技术。在相对定量中创建可靠数据的一个关键因素是对感兴趣基因的表达数据进行标准化。因此，所需的标准化因子是根据在特定样本材料中稳定表达的共扩增基因（即所谓的参考基因）的表达数据计算得出的。在这项研究中，我们展示了使用非模式物种验证潜在参考基因的重要过程。由于几乎没有已知的 Pallid Atlantic Forest Rat（Delomys sublineatus）序列，这种啮齿动物被用作濒危巴西雨林保护研究中的指示物种，因此比在模式物种中更难找到合适的引物。在为完全测序的 Mus musculus 设计的 9 个测试引物中，有 5 个可以用于建立适当的 SYBR-Green 测定法，并验证其恒定表达。使用三种不同的验证软件程序（BestKeeper、NormFinder 和 geNorm）分析了 Delomys 肝脏的 12 个 cDNA 的 qRT-PCR 结果。我们的方法表明，在这 5 个（Sdha、Canx、Pgk1、Actb 和 Actg1）潜在的参考基因中，前 4 个应在进一步的相对定量分析中用于准确标准化。将数据从附近的模式生物转移过来，使得高灵敏度实时 RT-PCR 甚至适用于自由生活的非模式生物。我们的方法可能适用于其他非模式生物。
