Zinzow-Kramer Wendy M, Horton Brent M, Maney Donna L
Department of Psychology, Emory University, Atlanta, GA, USA.
Department of Psychology, Emory University, Atlanta, GA, USA.
Horm Behav. 2014 Jul;66(2):267-75. doi: 10.1016/j.yhbeh.2014.04.011. Epub 2014 Apr 26.
Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.
定量实时聚合酶链反应（qPCR）正成为一种用于量化鸣禽大脑和内分泌组织中基因表达的常用工具。qPCR数据的准确分析依赖于选择合适的内参基因进行标准化，但关于鸣禽的论文中很少有内参基因验证的证据。在此，我们评估了10个潜在内参基因（18S、ACTB、GAPDH、HMBS、HPRT、PPIA、RPL4、RPL32、TFRC和UBC）在两种鸣禽（斑胸草雀和白喉带鹀）的大脑、垂体、卵巢和睾丸中的表达情况。我们使用geNorm和NormFinder两种算法来评估这些内参基因在我们样本中的稳定性。我们发现，一些在哺乳动物中用于目标基因标准化的最常用内参基因，如18S，其适用性高度依赖于组织类型。因此，它们并非这些鸣禽大脑和性腺的最佳选择。相比之下，我们鉴定出了替代基因，如HPRT、RPL4和PPIA，在这些物种的大脑、垂体和性腺中高度稳定。我们的结果表明，哺乳动物内参基因的验证不一定能外推到其他分类群。对于希望鉴定和评估鸣禽qPCR合适内参基因的研究人员来说，我们的结果应作为一个起点，并有助于提高鸣禽模型在行为神经内分泌学中的效能和实用性。
