Two-color Dye-swap DNA Microarray approach toward confident gene expression profiling in PMCAO mouse model for ischemia-related and PACAP38-influenced genes.

Toward twin goals of identifying molecular factors in brain injured by ischemic stroke, and the effects of neuropeptide pituitary adenylate-cyclase activating polypeptide (PACAP) on the ischemic brain, we have established the permanent middle cerebral artery occlusion (PMCAO) mouse model and utilized the Agilent mouse whole genome 4 × 44 K DNA chip. PACAP38 (1 pmol) injection was given intracerebroventrically in comparison to a control saline (0.9% NaCl) injection, to screen genes responsive to PACAP38. Two sets of tissues were prepared, whole hemispheres (ischemic and non-ischemic) and infract core and penumbra regions at 6 and 24 h. In this study, we have detailed the experimental design and protocol used therein and explained the quality controls for the use of total RNA in the downstream DNA microarray experiment utilizing a two-color dye-swap approach for stringent and confident gene identification published in a series of papers by Hori and coworkers (Hori et al., 2012-2015).