Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury

Abstract

Relative quantification by normalization against a stably expressed reference gene is a widely used data analysis method in microarray and quantitative real-time polymerase chain reaction (qRT-PCR) platforms; however, recent evidence suggests that many commonly utilized reference genes are unstable in certain experimental systems and situations. The primary aim of this study, therefore, was to screen and identify stably expressed reference genes in a well-established rat model of vocal fold mucosal injury. We selected and evaluated the expression stability of nine candidate reference genes. Ablim1, Sptbn1, and Wrnip1 were identified as stably expressed in a model-specific microarray dataset and were further validated as suitable reference genes in an independent qRT-PCR experiment using 2(-DeltaCT) and pairwise comparison-based (geNorm) analyses. Parallel analysis of six commonly used reference genes identified Sdha as the only stably expressed candidate in this group. Sdha, Sptbn1, and the geometric mean of Sdha and Sptbn1 each provided accurate normalization of target gene Tgfb1; Gapdh, the least stable candidate gene in our dataset, provided inaccurate normalization and an invalid experimental result. The stable reference genes identified here are suitable for accurate normalization of target gene expression in vocal fold mucosal injury experiments.

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.

Figure 2. Effect of vocal fold mucosal injury on the expression of candidate reference genes

RNA was extracted from rat vocal fold mucosa 1-7 days post-injury and non-injury controls and qRT-PCR was performed for nine candidate reference genes: (A) Sdha, (B) Sptbn1, (C) Actb, (D) Ablim1, (E) Wrnip1, (F) Gapdh, (G) B2m, (H) Hprt and (I) Ywhaz. Gene expression levels were calculated using the 2^-ΔCT method and are presented as mean fold change ± standard error. n = 5 animals per time point. p-values reflect one-way ANOVA across all time points. Ctl, non-injury control; d, day post-injury; n.s., non-significant difference.

Figure 3. Pairwise comparison-based (geNorm) analysis of candidate reference genes

(A) Distribution of expression levels (cycle threshold values) for all samples and all candidate reference genes. Twenty-five data points are shown for each gene, inclusive of injury and non-injury experimental conditions. (B) Average expression stability of a basket containing all candidate reference genes during stepwise exclusion of the least stable gene. 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). (C) 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.

Figure 4. Relative expression of the target gene Tgfb1 is significantly influenced by reference gene stability following vocal fold mucosal injury

Tgfb1 expression was normalized against reference genes Gapdh, Sdha, Sptbn1 and the geometric mean of Sdha and Sptbn1. Gene expression levels were calculated using the 2^-ΔΔCT method and are presented as mean fold change ± standard error. n = 5 animals per time point. *, p < .01 versus both the non-injury control condition and Gapdh normalized condition at the same time point; d, day post-injury.