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DNA microarrays are powerful tools for global analysis of gene transcript expression. However, their high cost and the need for replication have limited their use. Here, we report a new stripping technique applicable to microarrays hybridized with cRNA with RNase H that is reproducible, leaving the DNA oligonucleotide probes intact and available for adding two additional uses. A Pearson correlation was used to assess the agreement between the first-round hybridization and the second- and third-round hybridizations. Significant correlations (R2, 0.9893 and 0.975; P < 0.001) were observed among virgin arrays and stripped arrays hybridized with the same sample. Additionally, statistical class comparison analysis globally indicated that there were essentially no differences detected following three hybridizations. Dye-swapped microarrays produced similar results. However, arrays stripped with RNase H exhibited decreased efficiency of hybridization signal with increasing use. In the present study, the oligonucleotide microarrays can be used three times.