Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Sci Rep. 2016 Oct 18;6:35595. doi: 10.1038/srep35595.
Cell growth and differentiation are often driven by subtle changes in gene expression. Many challenges still exist in detecting these changes, particularly in the context of a complex, developing tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) allows relatively high-throughput evaluation of multiple genes and developmental time points. Proper quantification of gene expression levels by qRT-PCR requires normalization to one or more reference genes. Traditionally, these genes have been selected based on their presumed "housekeeping" function, with the implicit assumption that they are stably expressed over the entire experimental set. However, this is rarely tested empirically. Here we describe the identification of novel reference genes for the mouse mammary gland based on their stable expression in published microarray datasets. We compared eight novel candidate reference genes (Arpc3, Clock, Ctbp1, Phf7, Prdx1, Sugp2, Taf11 and Usp7) to eight traditional ones (18S, Actb, Gapdh, Hmbs, Hprt, Rpl13a, Sdha and Tbp) and analysed all genes for stable expression in the mouse mammary gland from pre-puberty to adulthood using four different algorithms (GeNorm, DeltaCt, BestKeeper and NormFinder). Prdx1, Phf7 and Ctbp1 were validated as novel and reliable, tissue-specific reference genes that outperform traditional reference genes in qRT-PCR studies of postnatal mammary gland development.
细胞生长和分化通常是由基因表达的微妙变化驱动的。在检测这些变化时，仍然存在许多挑战，特别是在复杂的发育组织中。定量逆转录聚合酶链反应（qRT-PCR）允许相对高通量地评估多个基因和发育时间点。qRT-PCR 对基因表达水平的适当定量需要以一个或多个参考基因进行归一化。传统上，这些基因是根据它们假定的“管家”功能选择的，隐含的假设是它们在整个实验过程中稳定表达。然而，这很少被经验性地测试。在这里，我们根据发表的微阵列数据集，描述了基于其在小鼠乳腺中稳定表达的新型参考基因的鉴定。我们将八个新的候选参考基因（Arpc3、Clock、Ctbp1、Phf7、Prdx1、Sugp2、Taf11 和 Usp7）与八个传统参考基因（18S、Actb、Gapdh、Hmbs、Hprt、Rpl13a、Sdha 和 Tbp）进行了比较，并使用四种不同的算法（GeNorm、DeltaCt、BestKeeper 和 NormFinder）分析了所有基因在小鼠乳腺从青春期前到成年期的稳定表达。Prdx1、Phf7 和 Ctbp1 被验证为新型可靠的组织特异性参考基因，在研究产后乳腺发育的 qRT-PCR 中优于传统参考基因。
