Dataset S3

GOTerm and Kegg Pathway analyses for probes differentially expressed by age

Dataset S5

Intersection of 36 genes differentially expressed in the cerebral cortex with Mammalian Genome Informatics Mammalian Phenotype data

Dataset S1

Probes significantly associated with age

Immunohistochemistry evidence for expression of ‘immunity-related’ genes in neurons and glial cells.

<p>Immunohistochemistry showing the protein expression of C1Q, NP2 and HLA-E in nondiseased human glial cells and neurons. Arrows denote the location of microglia (M) and neurons (N). Images A–B show C1Q staining in frozen temporal lobe sections of two adults. Images C–D show C1Q staining of microglia and neurons, respectively, in frozen temporal lobe sections of a child. Image E shows NP2 staining of microglia and neurons in frozen temporal lobe sections of an adult. Image F is a negative control of the adult frozen temporal lobe tissue. Images G–H show NP2 staining of microglia and neurons, respectively, in paraffin embedded temporal lobe section of a child. Images I–J show HLA-E staining of both microglia and neurons in frozen temporal lobe sections of two adults. Image K shows HLA-E staining of microglia and neurons in a frozen temporal lobe section of a child. Image L is a negative control of the frozen temporal lobe section of the child. We found evidence for the expression of all three proteins in both cell types of the child. However, in the adult, NP2 and HLA-E were present in both cell types but there was no evidence of C1Q expression.</p

Gene Ontology and KEGG pathway analyses

<p>(<b>reference  =  all genes</b>)<b>.</b> Gene Ontology Biological Process (GO_BP) and KEGG pathway analyses for probes with greater variance in childhood than in adulthood using as reference all genes called present on the array. The <i>expected</i> number of genes is the number of genes predicted for this term by random chance. The <i>observed</i> number of genes is the number of genes actually present in our dataset for this term. For example, in this context we would expect by random chance to see 13 genes annotated to the GO_BP term ‘immune system process’ (GO:0002376). Instead, we observed 75 genes annotated to this term (pFDR  = 0). The steepness of the slope of each line reflects statistical significance with steeper lines having smaller pFDR values. Those categories with the greatest slope (pFDR  = ≤0.02) are labeled in this figure. All 339 GO_BP terms and 19 KEGG pathways that met our enrichment criterion of pFDR ≤0.1 can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037714#pone.0037714.s005" target="_blank">Dataset S2</a>. Additional GO (Molecular Process and Cellular Component) data and plots can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037714#pone.0037714.s005" target="_blank">Dataset S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037714#pone.0037714.s001" target="_blank">Figure S1</a> respectively.</p

Paired differences between the standard deviation of genes in children vs. adults.

<p>The boxplot is made from for all 1095 probes with highest variance across all samples. Positive values indicate larger standard deviation (SD) in the younger group (children<15 years), whereas negative values indicate larger standard deviation in the older group (adults≥15 years). The median (heavy black line) represents the point at which 50% of the data are greater than (above the line) or less than (below the line) this value. The upper quartile (open box above the median) represents the 25% of the data greater than the median. The lower quartile (open box below the median) represents the 25% of the data less than the median. Note that 71% of the probes have greater standard deviation in the younger group. The maximum (above the upper quartile) and minimum (below the lower quartile) values excluding outliers are also shown. Outliers are drawn as open circles.</p

Test of the correlation between microarray and qPCR data.

<p>Y-axis shows mRNA expression levels [–DCt values (Ct reference–Ct target)] derived from qPCR experiments whereas the X-axis shows the log2 normalized microarray expression signal intensities. Correlations were considered significant when p<0.05. ρ = correlation coefficient.</p

Gene Ontology and KEGG pathway analyses

<p>(<b>reference  = 1095 probes with greatest variance</b>)<b>.</b> GO and KEGG pathway analyses for probes with greater variance in childhood than in adulthood using as reference the 1095 (∼5%) genes with highest variance across all samples. The <i>expected</i> number of genes is the number of genes predicted for this term by random chance. The <i>observed</i> number of genes is the number of genes actually present in our dataset for this term. For example, in this context we would expect by random chance to see 47 genes annotated to the GO_BP term ‘immune system process’ (GO:0002376). Instead, we observed 84 genes annotated to this term (pFDR  = 0). The steepness of the slope of each line reflects statistical significance with steeper lines having smaller pFDR values. Those categories with the greatest slope (pFDR  = ≤0.02) are labeled in this figure. All 10 GO_BP terms and 6 KEGG pathways that met our enrichment criterion of pFDR ≤0.1 can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037714#pone.0037714.s006" target="_blank">Dataset S3</a>. Molecular Function (MF) analyses did not meet enrichment criteria. Cellular Compartment (CC) has one term that met our criterion (MHC protein complex; pFDR  = 0.09; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037714#pone.0037714.s006" target="_blank">Dataset S3</a>).</p