PMC:1186732 / 51841-56370 JSONTXT

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In two of the three replicates, the mutant probe was labeled with Cy3 and the wild type with Cy5, and in the third replicate the dyes were swapped. Labeled probe was hybridized to microarray slides spotted with approximately 11,500 cDNA clones from the brain molecular anatomy project library (kind gift of B. Soares, University of Iowa; see http://trans.nih.gov/bmap/index.htm for details) and 500 cDNA clones from our lab collection. Slides were printed and hybridized as described [59,60]. After hybridization and washing of slides according to the manufacturer's instructions (Genisphere), the slides were scanned on an Axon Instruments (Union City, California, United States) GenePix 4000 scanner and images were analyzed using the accompanying GenePix Pro software package. The complete raw cDNA microarray data set are available in Tables S6–S14.\nTwo types of normalization were performed on the raw intensity scores derived from the GenePix Pro analysis. First, for a given experiment, the average intensity of all the spots in the weaker of the two channels (Cy3 or Cy5) was normalized to those in the stronger channel. Second, in a given set of experiments done in triplicate at a particular time point, the two experiments with the weaker average signal intensity over all spots were normalized to those in the third microarray with the strongest average signal intensity. All spots with signal levels equal to or below background were removed from the analysis. The resulting files contained on average about 6,000 spots. These files were then sorted according to Cy3/Cy5 signal intensity, and those spots with the 10% highest and 10% lowest intensity ratios (approximately 600 spots/experiment) were compared across the three experiments at a given time point using custom Perl scripts (available upon request from JCC). All spots which were present in the top 10% most up- or down-regulated genes in two out of three or three out of three experiments were recorded (the latter are listed in Table S3).\nMicroarray analysis of the P21 retinas was performed on Affymetrix mouse genome 430 2.0 GeneChip array (Affymetrix, Santa Clara, California, United States). A total of six microarray hybridizations were performed: three with probes derived from mutant RNA and three from wild-type. Probes were synthesized starting with 10 μg of total RNA for each sample according to manufacturer's instructions (Affymetrix). Hybridization, washing, and scanning of the microarrays were all performed at the Bauer Center for Genomics Research at Harvard University according to manufacturer's instructions (Affymetrix). Initial data analysis was carried out using the GeneChip Operating System (GCOS) software from Affymetrix. Pairwise comparisons were made between individual mutant microarray results and controls. All genes were removed from the analysis for which “absent” calls were made by the software for both the wild-type and mutant samples being compared. The remaining gene lists contained approximately 26,000 transcripts. These lists were then sorted according to the mutant-to-wild-type “signal log ratio” in order to identify the most markedly up- and down-regulated genes. The top 500 most up- and down-regulated transcripts (approximately 2% of all genes in each case) from each of the three pairwise comparisons between mutant and wild-type were compared using custom Perl scripts (available upon request from JCC) to identify those genes that were present in two or three out of three lists. Those genes that were up- or down-regulated in three out of three experiments were recorded (Tables S4 and S5). The complete pairwise Affymetrix microarray datasets are available in Tables 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