Annotation and analysis of 10,000 expressed sequence tags from developing mouse eye and adult retina.

BACKGROUND: As a biomarker of cellular activities, the transcriptome of a specific tissue or cell type during development and disease is of great biomedical interest. We have generated and analyzed 10,000 expressed sequence tags (ESTs) from three mouse eye tissue cDNA libraries: embryonic day 15.5 (M15E) eye, postnatal day 2 (M2PN) eye and adult retina (MRA). RESULTS: Annotation of 8,633 non-mitochondrial and non-ribosomal high-quality ESTs revealed that 57% of the sequences represent known genes and 43% are unknown or novel ESTs, with M15E having the highest percentage of novel ESTs. Of these, 2,361 ESTs correspond to 747 unique genes and the remaining 6,272 are represented only once. Phototransduction genes are preferentially identified in MRA, whereas transcripts for cell structure and regulatory proteins are highly expressed in the developing eye. Map locations of human orthologs of known genes uncovered a high density of ocular genes on chromosome 17, and identified 277 genes in the critical regions of 37 retinal disease loci. In silico expression profiling identified 210 genes and/or ESTs over-expressed in the eye; of these, more than 26 are known to have vital retinal function. Comparisons between libraries provided a list of temporally regulated genes and/or ESTs. A few of these were validated by qRT-PCR analysis. CONCLUSIONS: Our studies present a large number of potentially interesting genes for biological investigation, and the annotated EST set provides a useful resource for microarray and functional genomic studies.

Evaluation and optimization of procedures for target labeling and hybridization of cDNA microarrays.

PURPOSE: To evaluate and optimize methods of target labeling and microarray hybridization using eye gene microarrays. Standardized protocols that consistently produce low background and high intensity hybridization with small amounts of starting RNA are needed to extract differentially expressed genes from a pool of thousands of unaltered genes. METHODS: Two identical aliquots of RNA from P19 cell line were labeled with Cy3 or Cy5 dyes using four different methods and self-against-self hybridization was performed on mouse eye gene arrays. The validity and reproducibility of these protocols were further examined using target RNAs isolated from wild-type or neural retinal leucine zipper (Nrl) knockout mouse retinas. Hybridizations were also carried out on human gene array slides with different amounts of starting RNA from human retina. RESULTS: Using self-against-self hybridization, we optimized the protocols for direct labeling (R-square = 0.93), aminoallyl indirect labeling (R-square = 0.97), Genisphere 3DNA labeling (R-square = 0.96), and for microarray hybridization and washing. Although small amounts of initial RNA can be used in TSA method, inconsistent labeling was encountered under our experimental conditions. When retinal RNA targets from Nrl+/+ and Nrl-/- mice were tested by direct and aminoallyl indirect labeling protocols, both produced varying hybridization results with low intensity spots and non-uniform backgrounds. However, the Genisphere 3DNA labeling procedure consistently yielded strong hybridization and R-square values of 0.92 or higher. Furthermore, expression profiles were compatible with prior knowledge of this mouse model. Serial analysis of hybridizations with various starting amounts of RNA showed that the Genisphere 3DNA protocol could produce reliable signal intensity with 3 microgram of total RNA. CONCLUSIONS: We have systematically evaluated and optimized methods for target labeling, microarray hybridization and washing. These procedures have been used for expression profiling with 3 microgram of starting RNA. Our studies should encourage further use of microarray technology for gene profiling during eye development and in retinal diseases.

Gene expression profile of native human retinal pigment epithelium.

PURPOSE: To generate a profile of genes expressed in the native human retinal pigment epithelium and identify candidate genes for retinal and macular diseases. METHODS: Two cDNA libraries (one amplified, the other unamplified) were constructed using RNA isolated from native human RPE sheets. The sequence from the 5' end was obtained for randomly selected clones from the two libraries. Of these, more than 2000 expressed sequence tags (ESTs) were analyzed for similarity to sequences and gene clusters in public databases. RESULTS: EST analysis revealed several known RPE-expressed genes and more than 500 genes that have been characterized previously but were not known to be expressed in the RPE. Transthyretin and 90-kDa heat shock protein represent the most abundant transcripts identified in these RPE libraries. More than 200 novel ESTs and putative proteins were identified. An additional 344 sequences matched only the human genomic sequence. CONCLUSIONS: High-complexity cDNA libraries were generated from native human RPE. Analysis of ESTs generated from these libraries has yielded a profile of genes expressed in the native RPE. Several of the identified genes are known to play a significant role in the RPE. Novel ESTs, putative proteins, and genomic hits may represent as yet unidentified RPE-expressed genes and many of these, mapping in the region of retinal disease loci, may serve as candidate genes. In addition, the nonredundant set of more than 1100 genes and ESTs described herein will be a valuable resource for generating gene microarrays, which can assist in delineating RPE expression profiles during human disease pathogenesis.