Male 13-week-old ddY mice (Japan SLC Co., Hamamatsu, Japan) weighing 38-43 g were used. Dose of 25 mg/kg thioridazine HCl (Sigma-Aldrich Co) was used in the study. The drug was dissolved in acetic anhydride and diluted with 0.9% saline, resulting in a final concentration of acetic acid of 0.5%. The drugs were injected intraperitoneally once daily for 28 consecutive days in a volume of 0.1 ml/10 g body weight. Microarray experiments were performed using an Agilent G4121A Mouse Oligo Microarray Kit as per the manufacturer’s instructions. The frontal cortex was immediately removed and the RNA was stabilized in RNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA) and stored at -80°C until use. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). The RNA was amplified and labeled by the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). To synthesize cDNA, 200 ng total RNA was used. Vehicle-injected controls were labeled by cyanine 3 (PerkinElmer Life Sciences, Inc., Boston, MA) and drug-injected mice were labeled by cyanine 5. Hybridizations to the microarray were performed using the In situ Hybridization Kit Plus (Agilent). Doses of 750 ng cyanine 3-labeled cRNA, 750 ng cyanine 5-labeled cRNA, and control targets were mixed and fragmented in the kit’s fragmentation buffer, and then hybridized to the microarrays for 17 hours at 60°C in a hybridization rotator (Agilent) set to rotate at 4 rpm. Microarrays were washed in 6×SSC with 0.005% Triton X-102 at RT for 10 min, followed by 0.1×SSC with 0.005% Triton X-102 at 4°C for 5 min. The slides were dried, and then scanned by the Agilent G2565BA Microarray Scanner System. Data were analyzed using the Agilent Feature Extraction Software version 7.1. A rank consistency filter and LOWESS were used for dye normalization. Control mice and drug-injected mice were processed in parallel.
Keywords = thioridazine Lot batch = US22502606_16011978017256
