Confirmation of microarray results by sqRT-PCR.

<p>Beta-actin (<i>Bact</i>), a housekeeping gene, was used to normalize gene expression in mouse CH, RPE and PR. The light blue bars indicate expression levels in CH, the blue bars expression levels in the RPE and the dark blue bars indicate expression levels in PR. Similar to the microarray data the expression level is highest in the RPE and lowest in the PR. The sqRT-PCR results confirm our findings; however <i>Tshr</i> and <i>Slc16a8</i> show expression lower in RPE compared to choroid. Overall, the sqRT-PCR confirmation rate in this, and in all our previous studies (combined), using exactly the same methodology and platform to investigate neuroepithelia from human donor eyes and brains was 87% [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref012" target="_blank">12</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref014" target="_blank">14</a>].</p

Strategy to select RPE signature genes.

<p>In the first step of this strategy we laser dissect the RPE (and its adjacent layers, the CH and PR) for specific tissue collection. In the second step we statistically correct for possible contamination by adjacent layers.</p

Overview of the major biological functions found in a functional annotation by Ingenuity of the “Mouse High RPE gene expression” and “Human High RPE gene expression” datasets.

<p>The p-value for these categories are indicated as a range because each category contains sub-functions that have their own p-value.</p><p>Overview of the major biological functions found in a functional annotation by Ingenuity of the “Mouse High RPE gene expression” and “Human High RPE gene expression” datasets.</p

Most significant canonical pathways identified by Ingenuity for the “Mouse High RPE gene expression” and “Human High RPE expression gene expression” datasets.

<p>The left y-axis displays the–log of Benjamini-Hochberg corrected p-value. The right y-axis displays the ratio of the number of genes derived from our dataset, divided by the total number of genes in the pathway. The blue line indicates the threshold of the BH corrected p-value of 0.1.</p

The 22 signature genes that are specifically expressed in both RPE in mouse and in human.

<p>Derived from a comparison between our “Mouse RPE signature genes” dataset (this study) and two (modified) studies on the human RPE transcriptome [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref015" target="_blank">15</a>]. We show the gene symbol, genbank ID for both species and the GO annotation of each gene.</p><p>The 22 signature genes that are specifically expressed in both RPE in mouse and in human.</p

Strategy to determine “Interspecies RPE signature genes”.

<p>Schematic overview of our comparison strategy: our “Mouse RPE signature genes” dataset and “Human RPE signature genes” dataset, which contains (a modification of) two human RPE transcriptome datasets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141597#pone.0141597.ref015" target="_blank">15</a>]. This resulted in a new dataset, “Interspecies RPE signature genes”.</p

This diagram depicts the tight junction gene expression of mouse and human RPE, divided in four categories: high expression (>90^th percentile), moderate (50-90^th percentile), low (10-50^th percentile) and very low (<10^th percentile).

<p>On the x-axis the four categories are displayed and on the y-axis the amount of genes found in a category is depicted. Light blue circles contain genes expressed in mouse RPE, Dark blue circles genes expressed in human RPE. Genes inside the overlapping parts of the circles are expressed in the RPE in both species in that category.</p

Overview of significant canonical pathways assigned by the Ingenuity knowledge database to the 101 genes that are the result of comparing the “Mouse high RPE gene expression” and the “Human very low RPE gene expression” datasets.

<p>Overview of significant canonical pathways assigned by the Ingenuity knowledge database to the 101 genes that are the result of comparing the “Mouse high RPE gene expression” and the “Human very low RPE gene expression” datasets.</p

Our “Mouse RPE signature genes” dataset: 64 mouse RPE genes with an average expression of at least 2.5 fold higher in the mouse RPE than in both the PR and the CH with an adjusted p-value smaller than 0.01.

<p>Our “Mouse RPE signature genes” dataset: 64 mouse RPE genes with an average expression of at least 2.5 fold higher in the mouse RPE than in both the PR and the CH with an adjusted p-value smaller than 0.01.</p

Mouse specifically expressed CPE genes compared to human CPE.

<p>Mouse specifically expressed CPE genes compared to human CPE.</p