Media controls consistently failed to exhibit significant cDNA smears. To further evaluate the quality of the single cell cDNA mixtures, gene specific RT-PCR was performed using three genes known to be highly expressed in the developing retina. Robust bands were detected in those preparations that displayed the most robust cDNA smears and bands were routinely not observed with the media controls. One final, more comprehensive, approach was utilized to assess the single cell cDNAs. Ten micrograms of cDNA were labeled with Cy5 and hybridized to cDNA microarrays spotted in our laboratory. These microarrays contained,Ganoderenic-acid-D 12,000 ESTs derived from the retina and many retinal expressed genes from our laboratory. Many of the transcripts spotted on these cDNA microarrays showed significant signal when hybridized with cDNA from the single cells, whereas amplifications from media controls did not show signals above background. Taken together, these data demonstrated that more than 50% of isolated single retinal cells yielded cDNA of sufficient quantity and quality for more complete gene expression profiling on Affymetrix microarrays. Ten micrograms of cDNA from each single cell to be profiled was DNase treated,Ganoderic-acid-D labeled with biotinylated ddATP using TdT, and hybridized to Affymetrix mouse 430 2.0 oligonucleotide arrays using standard Affymetrix protocols. Since previous work on these genes did not assess retinal expression, it was not clear whether these signals were due to transcriptional activity of these loci in the retina, or were false positives due to the single cell method. Examination of SAGE tags for these genes showed that expression was detected for 2 of these 5 genes, suggesting that at least in these cases, there was bona fide retinal expression. To further assess the robustness of the single cell data, the levels of housekeeping genes were examined. It was not clear which genes should be used for this test, as several studies have demonstrated that housekeeping gene expression is highly variable, as assayed by microarray, SAGE, or other profiling methods. Similar variability has been observed using preparations of retinal tissue.