strain: Sprague-Dawley rat tissue: cerebellum age: 1 day old, neonatal treatment: nicotine (1 uM)
Treatment protocol
After 2 days in culture, nicotine, acetamiprid, or imidacloprid was added to the cell cultures at a final concentration of 1 μM. Nicotine, acetamiprid, and imidacloprid were dissolved in dimethyl sulfoxide, and their stock solutions (100 mM) were frozen at -30°C immediately prior to use. To exclude effects of vehicle, dimethyl sulfoxide (0.001% final) was added to the media for the control cultures and each of the treatment cultures. One-half of the culture medium was replaced with a fresh medium every 2 days for the 16 days in culture. During medium replacement, nicotine, acetamiprid, or imidacloprid was added to the culture at a final concentration of 1 μM.
Growth protocol
First, the cerebellar cells were cultured in a synthetic culture medium containing 1% fetal bovine serum for 8 days. After then, the neuron-enriched cultures were maintained in a serum-free synthetic medium consisted of Dulbecco's modified Eagle medium/F12 (Gibco, Thermo Fisher Scientific) with 10 μg/ml bovine insulin (Sigma-Aldrich), 100 μg/ml transferrin (Sigma-Aldrich), 30 nM sodium selenite, 5 nM thyroxine, 100 μM putrescine (Sigma-Aldrich), and antibiotics in a humidified incubator with 5% CO2/95% air, at 37°C.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cerebellar neuron-enriched cultures at 16 days in vitro using TRizol reagent (Invitrogen, Thermo Fisher Scientific) and Direct-zol RNA Miniprep Kit (ZYMO Research, Irvine, CA, USA) following manufacturer's recommendations. The RNA extracts were analyzed by Nanodrop 2000 (Thermo Fisher Scientific) and RNA quality was determined by the ratios A260/A280 (close to 2) and A260/A230 (more than 2). The quality of RNA was further assessed with a 2100 Bioanalyzer (Agilent Technologies, Valencia, CA, USA).
Label
Cy3
Label protocol
cRNA was synthesized and labelled using a Low input Quick Amp Labelling Kit (Agilent Technologies)
Hybridization protocol
Purified and labeled cRNA was hybridized to Whole Rat Gene Expression ver.3 4x44k Microarray (Agilent Technologies), according to the manufacturer's procedures. Hybridizations were performed for 17 hours at 65°C at 10 rpm in a rotating hybridization oven.
Scan protocol
The hybridized microarray slides were washed and scanned using Agilent SureScan microarray scanner using a scan resolution of 5 μm and dye channel of Green with PMT set to 100%.
Description
Gene expression in nicotine-treated cerebellar neuron-enriched culture of neonatal rats at 16 days in vitro
Data processing
Data was obtained using the Agilent Feature Extraction software (v 11.0.1.1 or 11.5.1.1) with set to default for all parameters. After then, quantile normalization and subsequent data processing were performed with using the GeneSpring GX v13.1 software package (Agilent Technologies). To determine the specific effects of nicotine, acetamiprid, and imidacloprid, raw data of each treatment and control were processed and the results were compared by GeneSpring. This semi-series were for nicotine treatment, and raw data of control was same as other semi-series of acetamiprid and imidacloprid. For each probe, the median of the log summarized values from all the samples was calculated and subtracted from each of the samples to get a transformed baseline.
Alteration of gene expression profile of cerebelllar neuron-enriched cultures from neonatal rats by nicotine, acetamiprid, or imidacloprid treatment [nicotine]
