From conventional relative gene expression analyses (Northern blotting, in situ hybridization, and RT-PCR), it has been reported that the expression of control genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin, used as references may be affected by ischemia. Therefore, we extended searching and evaluation at the mRNA level of transcripts whose expression levels were not changed by cerebral ischemia, using a high-density oligonucleotide array and statistical analysis in a rat global cerebral ischemia and reperfusion model. We added a hyperthermic factor and localization factor to ischemia and identified transcripts with a stable expression level under conditions even more disadvantageous than ischemia only. Screening of more than 8,000 transcripts with the Rat Genome U34A array yielded 28 transcripts, which we listed and classified according to their expression level. Widely used control genes, GAPDH and beta-actin, were not included, although cyclophilin A was included. In addition, we conducted a functional classification based on gene ontology. Under the functional classification of the 28 transcripts, many genes tended to be associated with metabolism. In conclusion, use of several transcripts is recommended, such as those we identified, as references in the analysis of gene expression in pathological models of ischemia. (C) 2004 Wiley-Liss, Inc.