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Purpose/Aim: Many genes have been associated with primary open-angle glaucoma (POAG). Knowing exactly where they are expressed in the eye helps to unravel POAG pathology and to select optimal targets for intervention. We investigated whether RNA in situ hybridization (RNA-ISH) is a convenient technique to obtain detailed pan-ocular expression data of these genes. We tested this for four diverse candidate POAG genes, selected because of unclear ocular distribution ( F5 and Dusp1) and relevance for potential new therapies ( Tnf, Tgfβr3 ). Optn , a POAG gene with well-known ocular expression pattern served as control. Methods: We made a list of candidate glaucoma genes reported in genetic studies. A table of their ocular expression at the tissue level was compiled using publicly available microarray data (the ocular tissue database). To add cellular detail we performed RNA-ISH for Optn, Tnf, Tgfβr3, F5 , and Dusp1 on eyes of healthy, 2-month-old, pigmented, and albino mice. Results: Expression of the Optn control matched with published immunohistochemistry data. Ocular expression of Tnf was generally low, with patches of higher Tnf expression, superficially in the corneal epithelium. F5 had a restricted expression pattern with high expression in the nonpigmented ciliary body epithelium and moderate expression in the peripapillary region. Tgfβr3 and Dusp1 showed ubiquitous expression. Conclusions: RNA-ISH is a suitable technique to determine the ocular expression pattern of POAG genes, adding meaningful cellular detail to existing microarray expression data. For instance, the high expression of F5 in the nonpigmented ciliary body epithelium suggests a role of this gene in aqueous humor dynamics and intraocular pressure. In addition, the ubiquitous expression of Tgfβr3 has implications for designing TGF-β-related glaucoma therapies, with respect to side effects. Creating pan-ocular expression maps of POAG genes with RNA-ISH will help to identify POAG pathways in specific cell types and to select targets for drug development.