Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain

Abstract

Background: Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods.We recently demonstrated that relative gene expression from qRT-PCR data normalized using GAPDH, ALG9 and RPL13A expression values mirrored relative expression using quantile normalization in Robust Multichip Analysis (RMA) on the Affymetrix GeneChip rhesus Macaque Genome Array.Having shown that qRT-PCR and Affymetrix GeneChip data from the same hormone replacement therapy (HRT) study yielded concordant results, we used quantile-normalized gene microarray data to identify the most stably expressed among probe sets for prospective internal reference genes across three brain regions from the HRT study and an additional study of normally menstruating rhesus macaques (cycle study). Gene selection was limited to 575 previously published human "housekeeping" genes. Twelve animals were used per study, and three brain regions were analyzed from each animal. Gene expression stabilities were determined using geNorm, NormFinder and BestKeeper software packages.

Results: Sequences co-annotated for ribosomal protein S27a (RPS27A), and ubiquitin were among the most stably expressed under all conditions and selection criteria used for both studies. Higher annotation quality on the human GeneChip facilitated more targeted analysis than could be accomplished using the rhesus GeneChip. In the cycle study, multiple probe sets annotated for actin, gamma 1 (ACTG1) showed high signal intensity and were among the most stably expressed.

Conclusions: Using gene microarray analysis, we identified genes showing high expression stability under various sex-steroid environments in different regions of the rhesus macaque brain. Use of quantile-normalized microarray gene expression values represents an improvement over traditional methods of selecting internal reference genes for PCR analysis.

Figure 1

Study methodology schematic. Flowchart outlining organization and groupings for analyses of data from the HRT and cycle studies. Alternating black and red text color is used for association of headings (left column) with appropriate rows of flowchart boxes. The six GABAergic pathway genes and three predicted normalizers for qRT-PCR are detailed in the methods. Numbered designations (1a, 1b... 2c) refer to separate selections of sequences to be analyzed (detailed in methods), with HRT sequences from 1a to 1c, and cycle study sequences from 2a to 2c. AMD = amygdala; E₂ = 17β-estradiol; E₂+P₄ = 17β-estradiol + progesterone; EF = early follicular (menstrual cycle phase); GABA = gamma-aminobutyric acid; HPC = hippocampus; LF = late follicular (menstrual cycle phase); MBH = arcuate nucleus of the medial basal hypothalamus; ML = mid luteal (menstrual cycle phase); OVX = ovariectomized; qRT-PCR = quantitative real-time polymerase chain reaction;

Figure 2

Relative gene expression stability in the rhesus macaque brain. GeNorm graphical output showing relative stability of genes expressed in three brain areas from three hormone replacement regimes. Stability values (M) are based on pairwise comparisons between the gene of choice and all other genes under consideration. The chart shows final stability values resulting from sequential elimination of the least stable genes after each set of pairwise analyses [34]. Therefore, the ordinate value shown for each gene represents the M-value obtained from the iteration before the gene was eliminated from analysis. Elimination sequence is shown from left to right. More stably expressed genes have lower M scores. Here the sample responsible for greatest influence on MBH variation was omitted from the analysis. When this sample was included the rank order of GAPDH and GAD1 were reversed.

Figure 3

NormFinder analysis for the five most stably expressed genes (qRT-PCR). Graphs show intra-group and inter-group variation [37] where gene expression quantity values were grouped according to brain region. Non-ordinate numbers show stability values for the given gene or gene combination.

Figure 4

NormFinder analysis for qRT-PCR prospective internal reference genes (qRT-PCR). Graphs show intra-group and inter-group variation [37] where gene expression quantity values were grouped according to brain region. Non-ordinate numbers show stability values for the given gene or gene combination.

Figure 5

Frequency distributions of relative expression stability values. Histograms show counts on the ordinate axis and expression stability values on the abscissa. Histograms are arranged according to study (first word) and analysis grouping scheme (second word). 1433 and 1444 probe set were used in the cycle and HRT studies respectively to represent genes likely to be reliably expressed under multiple conditions (housekeeping genes). Red lines show average expression stability values.

Figure 6

Relative expression stability of 'popular normalizers' in the cycle study. Relative expression stability of genes commonly used as internal references for normalization (popular normalizers). Each dot represents one probe set. Data from normally menstruating rhesus macaques. Values closer to zero (top of histogram) indicate greater expression stability. Microarray data from the amygdala, hippocampus and arcuate hypothalamus of normally menstruating rhesus macaques evaluated using the Affymetrix^® human GeneChip^®. The chart shows expression stabilities of 45 probe sets representing 15 popular genes. The ordinate axis shows expression stability rank relative to expression stabilities of 1433 probe sets representing 575 human genes reliably expressed under multiple conditions. Gene expression stability values were generated from gene expression values grouped according to brain region or menstrual cycle phase using NormFinder software.

Figure 7

Relative expression stability of 'popular normalizers' in the HRT study. Relative expression stability of genes commonly used as internal references for normalization (popular normalizers). Each dot represents one probe set. Data from ovariectomized rhesus macaques receiving hormone replacement. Values closer to zero (top of histogram) indicate greater expression stability. Microarray data from the amygdala, hippocampus and arcuate hypothalamus of ovariectomized rhesus macaques receiving E₂ or E₂+ P₄ and evaluated using the Affymetrix^® rhesus GeneChip^®. The chart shows expression stabilities of 39 probe sets representing 15 popular genes. The ordinate axis shows expression stability rank relative to expression stabilities of 1444 probe sets representing 575 human genes reliably expressed under multiple conditions. Gene expression stability values were generated from gene expression values grouped according to either brain region or treatment group using NormFinder software.