A new strategy to identify and annotate human RPE-specific gene expression.
BACKGROUND
To identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa.


METHODS
RPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44 k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software.


RESULTS
Our previous 22 k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways.


CONCLUSIONS
In this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE.
Booij
Judith C
JC
Department of Clinical and Molecular Ophthalmogenetics, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands.
ten Brink
Jacoline B
JB
Swagemakers
Sigrid M A
SM
Verkerk
Annemieke J M H
AJ
Essing
Anke H W
AH
van der Spek
Peter J
PJ
Bergen
Arthur A B
AA
D000368:Aged; D002829:Choroid / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D009928:Organ Specificity; D010786:Photoreceptor Cells / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction