Project description:Due to the lack of controlled microarray experimental data, evaluation of gene identification methods has been performed using simulated/manipulated data. As the ability of these approaches to mimic actual experimental data is questionable, conclusions drawn can be misleading. We applied spike-in approaches to develop experimental benchmark data and used it to evaluate various gene identification methods. <br> <br> mRNA was obtained from mouse ATCC CRL1606 hybridoma cells. From self hybridization results 200 genes that consistently exhibited a log ratio value close to 0 were randomly selected across a range of intensity. In-vitro transcripts of these 200 genes were obtained from their respective clones. From RNA gel electrophoresis and test array results, 169 of the 200 transcripts were deemed successful. Experiments were then conducted by spiking these 169 transcripts to the common pool of mRNA extracted from the hybridoma cells. With exception of the spiked in genes, the experimental set-up is identical to a self hybridization. This experiment is thus different from other microarray experiments as the set of differentially expressed genes is known as they have been artificially created. <br> <br> <br>
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
