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.