Sols-Morreale Biomedical Research Institute (IIBM), Spanish National Research Council (CSIC), Universidad Autónoma de Madrid (UAM), Madrid, Spain.
ABC-Ri, Algarve Biomedical Center Research Institute, Algarve Biomedical Center, Faro, Portugal.
Methods Mol Biol. 2025;2871:33-43. doi: 10.1007/978-1-0716-4217-7_4.
FOXO transcription factors belong to the forkhead protein family and are distinguished by their unique forkhead (FKH) DNA-binding domain. In the realm of mammals, four FOXO paralogs are recognized: FOXO1, FOXO3, FOXO4, and FOXO6. These paralogs are evolutionary counterparts of the daf-16 gene discovered in the nematode C. elegans. A key feature shared by these paralogs is a consensus binding site known as the DAF-16 family protein-binding site (DBE: 5'-TTGTTTAC-3'). The functional outcome of FOXO transcription factors primarily hinges on their affinity for these specific binding sites within the promoters of their target genes. Nevertheless, it is worth noting that many of these target genes exhibit tissue-specific expression patterns. Consequently, there is not a single FOXO target gene whose expression can reliably serve as a universal indicator of FOXO activity across all cell types and tissues or in response to all stimuli. In light of these considerations, we present a collection of target genes that, when collectively assessed, can accurately gauge FOXO activation. In this chapter, we outline a specific protocol for utilizing quantitative reverse transcription polymerase chain reaction (qRT-PCR) to measure the expression levels of these genes.
FOXO 转录因子属于叉头蛋白家族，其特征是具有独特的叉头（FKH）DNA 结合域。在哺乳动物中，有四个 FOXO 基因的同源物：FOXO1、FOXO3、FOXO4 和 FOXO6。这些同源物是在秀丽隐杆线虫中发现的 daf-16 基因的进化对应物。这些同源物的一个共同特征是存在一个称为 DAF-16 家族蛋白结合位点（DBE：5'-TTGTTTAC-3'）的保守结合位点。FOXO 转录因子的功能结果主要取决于它们与目标基因启动子中这些特定结合位点的亲和力。然而，值得注意的是，许多这些目标基因表现出组织特异性的表达模式。因此，不存在一个单一的 FOXO 目标基因，其表达可以可靠地作为所有细胞类型和组织中 FOXO 活性的通用指标，也不能作为对所有刺激的反应的通用指标。有鉴于此，我们提出了一组靶基因，当综合评估时，可以准确衡量 FOXO 的激活。在本章中，我们概述了一种利用定量逆转录聚合酶链反应（qRT-PCR）测量这些基因表达水平的具体方案。
