Changes in gene expression following induction of ischemic tolerance in rat brain: detection and verification

Tolerance against ischemic insults can be elicited in the CA1 region of rat hippocampus by inducing a short ischemic period 2-3 days prior to the ischemic insult. To detect genes whose expression changes following induction of ischemic tolerance (IT), we applied a differential display technique called restriction fragment differential display-PCR (RFDD-PCR). RFDD-PCR displays the coding region of mRNA and allows detection of differentially expressed mRNA. Double-stranded cDNA generated using a T25V primer is digested by the endonuclease TaqI, and adapters are ligated onto the cDNA fragments. When amplifying the adapter-containing cDNA fragments under high-stringency conditions, reproducible PCR profiles are obtained. By comparing these profiles from naïve and ischemia-tolerant rat brains statistically, significant expression changes of 20 fragments were identified. To verify the observed changes, quantitative PCR and in situ hybridization were performed for three fragments representing proteins with quite different functions (GluR2-flop, SC1, and p68 RNA helicase). Quantitative PCR displayed the same degree of regulation as RFDD-PCR, but in situ hybridization did not display any regulation. As the applied PCR-based techniques detect only polyadenylated mRNA, whereas in situ hybridization detects both nonadenylated and adenylated mRNA, changes in the polyadenylation state of the mRNA, rather than inconsistent changes in the total amount of mRNA, probably explain this discrepancy. Thus, our results show that the expression of genes hitherto not related to IT changes with the induction of IT and that the degree of regulation displayed by RFDD-PCR can be verified by quantitative PCR.