Microarray evaluation of the Listeria monocytogenes infection and amoxicillin treatment in mice.

By using Affymetrix Mouse Genome Arrays and 20 biological replicates per experimental condition, the predictive value of liver and blood gene expression profiles previously identified was validated as predictive of Listeria monocytogenes infection severity (lethal and nonlethal infection). The ability of these genes to predict the outcome of antibiotic treatment was also assessed. Lethally infected BALB/c mice were treated with amoxicillin at 10 or 20 mg/kg; only the higher dose prevented death. The liver genes predicted that 70% of the animals treated at 10 mg/kg, but only 25% of the mice treated at 20 mg/kg, belonged to the lethal infection group, and this prediction was similar to the ultimate mortality outcome. These results confirm the value of microarrays as tools to predict host response to infection and efficacy of antibacterial therapy. These results might lead to applications that would help clinicians to adjust antibiotic dosages for efficient treatment but yet without toxicity.

Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis.

Gene expression analysis has become an invaluable tool for understanding gene function and regulation. However, global expression analysis requires large RNA quantities or RNA preamplification. We describe an isothermal messenger RNA (mRNA) amplification method, Ribo-SPIA, which generates micrograms of labeled cDNA from 5 ng of total RNA in 1 day for analysis on arrays or by PCR quantification. Highly reproducible GeneChip array performance (R2 > 0.95) was achieved with independent reactions starting with 5-100 ng Universal Human Reference total RNA. Targets prepared by the Ribo-SPIA procedure (20 ng total RNA input) or the Affymetrix Standard Protocol (10 microg total RNA) perform similarly, as indicated by gene call concordance (86%) and good correlation of differential gene expression determination (R2 = 0.82). Accuracy of transcript representation in cDNA generated by the Ribo-SPIA procedure was also demonstrated by PCR quantification of 33 transcripts, comparing differential expression in amplified and nonamplified cDNA (R2 = 0.97 over a range of nearly 10(6) infold change). Thus Ribo-SPIA amplification of mRNA is rapid, robust, highly accurate and reproducible, and sensitive enough to allow quantification of very low abundance transcripts.