|Application:||Gene expression microarray analysis|
|Number of samples:||8|
|Release date:||May 1 2012|
|Last update date:||May 10 2018|
|Diseases:||Brain Ischemia, Hydranencephaly, Neoplasms|
|Chemicals:||Pituitary Adenylate Cyclase-Activating Polypeptide|
|Dataset link||Transcriptomics and Proteomics Analyses of the PACAP38 influenced Ischemic Brain in Permanent Middle Cerebral Artery Occlusion Model Mice|
The PMCAO model mice were generated as described previously. Briefly, the mice were anesthetized with 4% sevoflurane (induction) and 2% sevoflurane (maintenance) in a 30% O2 and 70% N2O gas mixture via a face mask. An incision was then made in the cervical skin followed by opening of the salivary gland, and the right common carotid artery was visualized. A midline cervical incision was made to expose the external carotid artery. The intraluminal filament technique was used, to generate the PMCAO model. - The PACAP38 (1 µl containing 1 pmol) or 1 µl of saline (0.9% NaCl) was injected intracerebroventrically, immediately after PMCAO. PACAP38 (Peptide Institute Inc., Osaka, Japan; supplier temperature was -20ºC and was stored at -80ºC) was dissolved in, and diluted ×10 times with 0.9% NaCl just before use. - In the sham control animals, following exposure of the external carotid artery, the wound was sutured. The PACAP38 or saline was injected intracerebroventrically in the same concentrations as above. After injections, the animals were returned to their cages. - A total of eight groups were prepared: four groups of three, seven, four and five mice in the PMCAO plus PACAP38 and PMCAO plus saline cohorts at 6 and 24 h after operation, respectively, and five mice each in the control (sham) plus PACAP and saline groups at 6 and 24 h after operation, respectively. - We used three mice each in PMCAO groups that exhibited neurological grades G1 and G2 and three mice each at random in sham groups for the subsequent downstream analysis. Six or 24 hours post-injection of PACAP38 or saline, the mice were removed from their cages, decapitated, and their brains carefully removed on ice. The left (contralateral) and right (ipsilateral; ischemic) hemispheres were dissected and placed in 2 mL Eppendorf tubes, which were then quickly immersed in liquid nitrogen before being stored in -80ºC prior to further analysis. - For analysis of gene components, the tissues were ground to a very fine powder with liquid nitrogen, used for total RNA extraction, quantity and quality check, followed by cDNA synthesis, and RT-PCR. A mouse 4 x 44K whole genome oligo DNA microarray chip (G4122F, Agilent Technologies, Palo Alto, CA, USA) was used for global gene expression analysis using the ipsilateral (ischemic) hemisphere. Briefly, total RNA (900 ng; 300 ng each replicate pooled together) was labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Fluorescently labeled targets of control (sham) as well as treated (PMCAO) samples with PACAP38 or without PACAP38 (saline) were hybridized to the same microarray slide with 60-mer probes. - Here, we compared the PMCAO plus PACAP38 injected mice to PMCAO plus saline, i.e. the ipsilateral brain region of the PMCAO mice was compared with the same right hemisphere of the control mice. Similarly, Sham control plus PACAP38 injected mice to Sham control plus saline were analyzed to identify only the PACAP38 influenced genes. A flip labeling (dye-swap or reverse labeling with Cy3 and Cy5 dyes) procedure was followed to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cDNA.