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Glass cDNA microarrays can be used to profile the expression of thousands of gene targets in a single experiment. However, the potential for hybridization cross-reactivity needs to be considered when interpreting the results. Here, we describe hybridization experiments with a model array representing four distinct functional classes (families): chemokines, cytochrome P-450 isozymes, G proteins, and proteases. The cDNA clones selected for this array exhibited pairwise sequence identities ranging from 55% to 100%, as determined by a homology scoring algorithm (LALIGN). Targets for microarraying were amplified by PCR and spotted in 4-fold replication for signal averaging. One designated target from each family was further amplified by PCR to incorporate a T7 promoter sequence for the production of synthetic RNA transcripts. These transcripts were used to generate fluorescent hybridization probes by reverse transcription at varying input concentrations. As expected, hybridization signals were highest at the matching target elements. Targets containing less than 80% sequence identity relative to the hybridization probe sequences showed cross-reactivities ranging from 0.6% to 12%. Targets containing greater than 80% identity showed higher cross-reactivities (26%-57%). These cross-reactive signals were analyzed for statistical correlation with the length of sequence overlap, percent sequence identity, and homology score determined by LALIGN. Overall, percent sequence identity was the best predictor of hybridization cross-reactivity. These results provide useful guidelines for interpreting glass cDNA microarray data.

Hybridization Cross-Reactivity within Homologous Gene Families on Glass cDNA Microarrays