Hybridization cross-reactivity within homologous gene families on glass cDNA microarrays

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