Top 30 genes significantly more highly expressed in the RPE compared to the IE.

<p>The genes that are in bold were shown to be enriched in the human RPE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182983#pone.0182983.ref026" target="_blank">26</a>]. Asterisks mark the genes that might be present in our dataset by contamination of the mRNA on the photoreceptor-RPE interface or may be expressed to some extent in both adjacent cell layers (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182983#sec021" target="_blank">Materials and methods</a>).</p

Top 20 significant canonical pathways of the core analysis in IPA (Ingenuity) of most highly expressed genes of the IE and the RPE.

<p>P-values indicate the significance of enrichment for the most highly expressed genes from our dataset. P-values were corrected for multiple testing using the Benjamini-Hochberg (B-H) false discovery rate. The upper graph (light blue bars) represents the–log(B-H) p-value of the RPE and the lower graph (dark blue bars) represents the–log(B-H) p-value of the IE. The orange line indicates the threshold of B-H corrected p<0.001.</p

Heatmap for the expression of genes related to RPE specific functions.

<p>The normalized expression data are converted to heat map color using the mean and maximum values for each gene. The intensity scale of the standardized expression values ranges from dark blue (low expression) to dark orange (high expression). We added a hierarchical cluster tree that shows that the IE samples cluster together and the RPE samples cluster together.</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

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

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

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

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

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

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