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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Gene expression profiling in mitochondrial disease: assessment of microarray accuracy by high-throughput Q-PCR.

Mitochondrial diseases are a heterogeneous array of disorders with a complex etiology. Use of microarrays as a tool to investigate complex human disease is increasingly common, however, a principle drawback of microarrays is their limited dynamic range, due to the poor quantification of weak signals. Although it is generally understood that low-intensity microarray 'spots' may be unreliable, there exists little documentation of their accuracy. Quantitative PCR (Q-PCR) is frequently used to validate microarray data, yet few Q-PCR validation studies have focused on the accuracy of low-intensity microarray signals. Hence, we have used Q-PCR to systematically assess microarray accuracy as a function of signal strength in a mouse model of mitochondrial disease, the superoxide dismutase 2 (SOD2) nullizygous mouse. We have focused on a unique category of data--spots with only one weak signal in a two-dye comparative hybridization--and show that such 'high-low' signal intensities are common for differentially expressed genes. This category of differential expression may be more important in mitochondrial disease in which there are often mosaic expression patterns due to the idiosyncratic distribution of mutant mtDNA in heteroplasmic individuals. Using RNA from the SOD2 mouse, we found that when spotted cDNA microarray data are filtered for quality (low variance between many technical replicates) and spot intensity (above a negative control threshold in both channels), there is an excellent quantitative concordance with Q-PCR (R2 = 0.94). The accuracy of gene expression ratios from low-intensity spots (R2 = 0.27) and 'high-low' spots (R2 = 0.32) is considerably lower. Our results should serve as guidelines for microarray interpretation and the selection of genes for validation in mitochondrial disorders.[1]


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