Microarray Gene Expression Analyses in Medaka (Oryzias Latipes) Exposed to Hypoxia

Microarray Gene Expression Analyses in Medaka (Oryzias latipes) Exposed to Hypoxia

Melissa C. Wells, Molecular Biosciences Research Group, Department of Chemistry and Biochemistry, Texas State University

Zhenlin Ju, Molecular Biosciences Research Group, Department of Chemistry and Biochemistry, Texas State University

Sheila J. Heater, Molecular Biosciences Research Group, Department of Chemistry and Biochemistry, Texas State University

Ronald B. Walter, Molecular Biosciences Research Group, Department of Chemistry and Biochemistry, Texas State University

We are investigating the genomic and proteomic effects of hypoxia exposure using the Japanese medaka (Oryzias latipes) aquaria fish model as a tool for biomarker discovery. We have developed a hypoxia exposure system allowing programmable exposure scenarios and have initiated experimental assessment of changes in gene expression and protein abundance using microarray and 2D-DIGE gel analyses of hypoxia exposed fish.

We present the design, construction, validation, and subsequent use of a medaka 8,046 (8K) unigene oligonucleotide microarray to begin the study of hypoxia exposure. Array performance was validated via self-self hybridization. Optimization of sample size needed for robust array data, based upon the number features detected and the signal intensity, suggest 2 µg total RNA as a starting template for amplification is sufficient. For treatment, adult medaka are exposed to a hypoxic environment of 4% dissolved oxygen (DO) for 2 days and then the DO lowered to 2% for an additional 5 days. Upon sacrifice, changes in gene expression in brain, liver, skin, and gill tissues of these fish were assessed in conjunction with matched control fish exposed similarly to 18% DO. Analyses of array results identified 501 features from brain, 442 from gill, and 715 features from liver that exhibit statistically significant changes in transcript abundance upon hypoxia exposure. Nine features were found to exhibit common expression patterns between all three tissues. Data mining of the array results suggest hypoxic exposure results in a general slowdown of metabolic function. Real-time PCR was then employed to support the microarray results and this independent validation agreed well with the microarray findings. Overall these results indicate the medaka microarray will be a sound diagnostic tool for changes in gene expression due to hypoxia exposure.