Figure 1. Microarray-based selection of candidate reference genes.

An expression microarray dataset was generated representing rat vocal fold mucosa 3, 14 and 60 days post-injury. Age-matched non-injury controls were included at each time point. A total of 23 arrays (14 post-injury, 9 non-injury controls) were included in the dataset. (A) Heat map showing log₂ fluorescent intensity for the 50 genes with the lowest coefficient of variation across all arrays representing both injury and non-injury experimental conditions. Genes are ranked by ascending coefficient of variation. Eight genes exhibited mean fluorescent intensity within the mid-90% of all genes on the arrays; these genes were subjected to further analysis. (B) Distribution of log₂ fluorescent intensity for the eight genes of interest identified in panel A. Twenty-three data points are shown for each gene, inclusive of injury and non-injury experimental conditions. (C) Average expression stability of a basket containing the eight genes of interest during stepwise exclusion of the least stable gene (geNorm algorithm). The abscissa reflects the number of genes under consideration throughout the stepwise procedure; the ordinate reflects the average expression stability (M-value) of the remaining genes after exclusion of the least stable gene (indicated by name on the plot). (D) Pairwise variation (V_i/V_i+1) between two sequential normalization factors (NF_i and NF_i+1) calculated to determine the optimal number of reference genes for accurate normalization (geNorm algorithm). Ctl, non-injury control; PI, post-injury; d, day.