Pictorial representation of microarray data.

<p>Microarray analysis of human healthy articular cartilage was conducted to assess the effect of age, BMI, and sex. <b>A.</b> Venn diagrams represent the number of differentially expressed genes in each comparison. Values in parentheses are the numbers of differentially expressed genes. Values in overlapping areas are the numbers of genes common to any two comparisons. <b>B.</b> Numbers of differentially expressed gene transcripts across all three factors are shown. Number of gene transcripts shown for age and BMI indicate positively (red) and negatively (green) correlated gene transcripts while for sex they represent up-regulated (red) and down-regulated (green) gene transcripts in females versus males. <b>C.</b> Hierarchical clustering map, representing the transcripts that were significantly (P<0.05) differentially expressed between the two sexes is shown. Each vertical row depicts a single sample, and each horizontal line stands for a single gene transcript. Green color indicates down-regulated gene transcripts while red color indicates up-regulated gene transcripts. <b>D.</b> The differentially expressed gene transcripts at all fold changes are shown in the form of volcano plots to show the trend of expression by P value (y axes) and fold change (x axes). Gene transcripts outside the thick black lines are those that have ≥1.5 fold expression x = gene transcripts with lowest P value (statistically highly significant) y = gene transcripts with highest fold change (biologically highly significant), green circles = gene transcripts down-regulated in females, red circles = gene transcripts up-regulated in females, N/C = no change.</p

Selected list of gene transcripts correlated with age and BMI^*.

<p>Selected list of gene transcripts correlated with age and BMI^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155373#t002fn001" target="_blank">*</a>}.</p

Heat map view of z-scored expression profiles of gene transcripts differentially expressed between healthy and OA cartilage.

<p>Hierarchical clustering separated the study patients into three distinct clusters. The list of gene transcripts used for cluster analysis was generated in a prior study by Karlsson et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155373#pone.0155373.ref040" target="_blank">40</a>] that looked at the transcriptome-wide RNA expression of healthy and OA cartilage. This list represented all the expressed gene transcripts differentially expressed between healthy and OA cartilage at >15-fold difference.</p

Gene transcripts common to age, BMI and sex^*.

<p>Gene transcripts common to age, BMI and sex^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155373#t005fn001" target="_blank">*</a>}.</p

Heat map view of z-scored expression profiles of genes associated with OA risk-alleles.

<p>Hierarchical clustering identified two clusters that have distinct expression pattern. When known, the expression pattern of OA-associated genes is provided in parentheses.</p

Biological processes associated with sex.

<p>Based on statistical significance i.e. -log (P value) as determined by IPA analysis, the biological processes elevated in females (A) and males (B) are shown.</p

Characteristics of study participants and quality of RNA samples.

<p>Characteristics of study participants and quality of RNA samples.</p

Findings from microfluidic digital PCR and comparison with microarray for selected gene transcripts.

<p>The expression of six differentially expressed gene transcripts was validated by microfluidic-based digital PCR. The expression profile of the entire set of gene transcripts obtained from TaqMan digital PCR assay was concordant with the microarray data. Pearson correlation coefficient was calculated for age (left panel; red microarray, blue digital PCR), and BMI (center panel; red microarray, blue digital PCR) and differential expression was presented for sex (right panel; these data are presented as mean with standard error of the mean). r = correlation coefficient</p

Biological processes common to BMI age and sex^*.

<p>Biological processes common to BMI age and sex^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155373#t006fn001" target="_blank">*</a>}.</p

Biological processes represented by differentially expressed gene transcripts by age, BMI, and sex.

<p>Biological processes represented by differentially expressed gene transcripts by age, BMI, and sex.</p