MDMA ((±)-3, 4-methylenedioxymethamphetamine hydrochloride, CAS # 92279-84-0, purity ≥99.9%) was obtained from the Reproductive and Development Toxicology Division of the Toxicological Research Department, The National Institute of Toxicological Research, Seoul, Korea. MDMA was dissolved in isotonic saline at doses of 20 mg/kg at volumes corresponding to 3ml/kg, and was administered orally (p.o.).
Growth protocol
Male and female C57BL/6N mice (6 weeks), purchased from the Orient Co. Ltd., were kept under SPF-conditions. All mice were acclimatized in the laboratory according to the ‘Guide for the Care and Use of Laboratory Animals’ (US NIH publication No. 86-23, 1985 Ed.)
Extracted molecule
total RNA
Extraction protocol
Brain regions were then dissected free, frozen on dry ice, and stored in a LN2 tank until further analysis. Total RNA was isolated from the brain tissue using the TRI reagent (Molecular Research Center, Cincinnati, OH.,USA), according to the manufacturer's instructions (Life Technology, Rockville, MD, USA). Briefly, the tissues were frozen in liquid nitrogen, pulverized, homogenized in 1 ml of TRI reagent, and incubated for 5 min at room temperature; 200 µl if chloroform was added, vigorously mixed, and incubated for 3 min at room temperature. The samples were centrifuged at 14000 rpm for 20 min, aqueous phases were transferred to fresh tubes with an equal volume of isopropanol, and incubated on ice for 30 min. After centrifugation at 14000 rpm for 15 min, RNA pellets were washed in 75% ethanol and dissolved in RNase-free water. RNA qualities were assessed using the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA), and concentrations were determined using a ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Samples with a minimum 28S/18S ratio of > 1.6 were selected for further study.
Label
Digoxigenin-UTP
Label protocol
1 microgram of total RNA was used to transcribe DIG labeled cRNA using Applied Biosystems Chemiluminescent RT-IVT kit v2.0
Hybridization protocol
Fifteen microgram of DIG labeled fragmented cRNA was used for microarray hybridization and performed according to the Applied Biosystems protocols
Scan protocol
Chemiluminescence detection, image acquisition, and analysis were performed using Applied Biosystems 1700 Chemiluminescent Microarray Analyzer, according to the manufacturer's instructions. Images were auto-gridded and chemiluminescent signals were quantified, corrected for background, and spatially normalized.
Description
N5_F1FCCX-5
Data processing
For primary data filtering, spots with a SNR (Signal to Noise Ratio) <1.5 were excluded, and the remaining filtered data were used for further analysis. Quantile normalization was used to normalize data. All process performed using GenPlex software (Istech Inc., Korea)
