Catherine W. Ernst, Valencia D. Rilington, Nancy E. Raney, Jianbo Yao, Sue S. Sipkovsky, Peter M. Saama, Robert J. Tempelman and Paul M. Coussens
Department of Animal Science and Center for Animal Functional Genomics, Michigan State University, East Lansing, MI 48824
The establishment of specialized cell types during development of specific tissues involves the expression of distinct sets of cell type-specific genes. A long-term objective in our laboratory is to identify differentially expressed genes in developing pig skeletal muscle. We have constructed a normalized porcine skeletal muscle cDNA library (PoSM) using poly (A)+RNA from hind limb muscle of pigs at 45 and 90 d of gestation, birth, 7 wk and 1 yr of age. Our database currently contains 3’- or 5’-end sequence for 782 PoSM clones. We have constructed a cDNA microarray containing 28 clones previously derived from differential display reverse transcription PCR experiments and 740 PoSM clones. All clones were spotted in triplicate and arrayed in 48 8X8 patches. A portion of the bacteriophage Lamba Q gene was spotted in the top left corner of every patch as a positive hybridization control. Total RNA from skeletal muscle of pigs at 60 d of gestation and 7 wk of age was labeled with both Cy3 and Cy5 in reciprocal experiments. The entire experiment was replicated so that a total of four microarray slides were screened. Fifty-three clones were overexpressed by at least 2-fold (40 by at least 2.5-fold) in 60-d fetal skeletal muscle as compared to 7-wk postnatal muscle in all four experiments. No clones were identified to be overexpressed in 7-wk postnatal muscle. Our results demonstrate that cDNA microarray technology provides a powerful and rapid means for identifying differentially expressed genes.
Keywords: pig, skeletal muscle, cDNA microarray