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X. Huang, L.D. Hazlett; Microarray and Real-Time RT-PCR Analysis of Gene Expression in Pseudomonas aeruginosa Corneal Infection in Mice . Invest. Ophthalmol. Vis. Sci. 2003;44(13):862.
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Purpose: This work compares the gene expression profile of normal (uninfected) vs. P. aeruginosa infected cornea of susceptible C57BL/6 (B6) (cornea perforates) vs. resistant BALB/c (cornea heals) mice using oligonucleotide microarray and confirms selected gene expression data using quantitative real-time RT-PCR. Methods: Corneas (left eye) were scarified and infected topically with P. aeruginosa (5 µl of 106 CFU/µl). At 1 day p.i., total RNA was isolated from the cornea (n =10 normal and 10 infected/group) using TRIzol. RNA concentration was spectrophotometrically determined and quality checked by Agilent Technologies 2100 Bioanalyzer. Total RNA was tested using Affymetrix microarray chips (12,000 murine genes) and average > 2-fold changes arbitrarily considered significant. Quantitative real-time RT-PCR (Cepheid Smart Cycler System) was used to confirm selected genes in each cluster generated by microarray testing. Fold differences of gene expression in normal vs. infected cornea for each group were calculated after normalizing to ß-Actin. Results: 1,097 regulated transcripts were detected and organized into nine different clusters by a self organizing map algorithm according to their different behaviors in each group of mice. Biological categorization revealed that the infected cornea in B6 mice showed a dominant type 1 immune response profile. Cytokines (e.g., IL-12, TNF-α, GM-CSF, Lymphotoxin and IFN-γ receptor) and chemokines (e.g., MIP-1α, MIP-1ß, RANTES, MIP-2, MCP-1, CCR-1, 2, 5, and 7) were up-regulated. In contrast, the infected cornea of BALB/c mice showed a dominant type 2 immune response with cytokines (e.g., IL-4, 5, 10, 13) and chemokines (e.g., CCR-4, 8, 10, MCP-2, TCA-3, and TARC) up-regulated. Additionally, the infected cornea of BALB/c mice showed increased gene expression of factors associated with matrix remodeling/tissue repair and/or bacterial killing (e.g., TIMP-2, MMP-11, EGF and iNOS). Data also revealed that expression of several genes that promote apoptosis (e.g., caspases-3, -8, -9 and cytochrome C) were up-regulated in the cornea of BALB/c mice, whereas genes with known apoptosis-inhibiting activity (e.g., BCL-2, apoptosis inhibitor 1 and gelsolin) were significantly up-regulated in the cornea of B6 mice. Conclusions: These results demonstrate that microarray analysis is a powerful approach for determining the complexity of transcriptional profiles that dictate susceptibility vs. resistance to bacterial infection and together with real-time RT-PCR confirmation may reveal novel target genes for therapeutic intervention.
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