Pons and medulla oblongata of one-day old SHR and WKY rats (n=20) were dissected out and dissociated in cold isotonic salt solution, pH 7.4. Cells were suspended in Neurobasal A media (Invitrogen) supplemented with L-glutamine (250umol/L, Sigma), glutamax (250umol/L, Gibco), B27 (2%, Gibco) e gentamicin (40mg/L, Gibco). Viable cells were counted and plated on poly-D-lysine-coated culture dishes (35mm, Nunclon, USA) at the concentration of 1800 cells/mm2. The experiment was repeated three times for each strain using different pools of animal tissue. Cultures were kept in a humidified incubator with 5%CO2 and 95% air, at 37ºC, for 9 days prior to mRNA extraction.
Biomaterial provider
Animal facilities of the Department of Physiology, Institute of Biosciences, University of São Paulo
Treatment protocol
Cultured cells (n=3 from each strain) were treated with 10μM of nicotine (Sigma), diluted in fresh culture medium, over 24 hours. Cell cultures kept for the same period of time in fresh medium without nicotine were used as a control. The experiment was repeated three times using different pools of animals. At the end of the treatment, cells from SHR or WKY animals, treated or untreated with nicotine, were lysed for RNA extraction.
Extracted molecule
total RNA
Extraction protocol
Cells from three dishes were pooled and total RNA was extracted according to the manufacturer instructions using the RNAspin Mini Kit (GE Healthcare, UK). The RNA was purified and treated with DNAse before the assessment of its concentration and quality by UV spectrophotometry and agarose gel electrophoresis, respectively.
Label
biotin-NTP
Label protocol
Target labeling and hybridization was performed strictly as recommended by the array manufacturer (GE Healthcare, UK). In brief, the purified and high-quality total RNA was used in reverse transcription reactions to convert messenger RNA (mRNA) in double-stranded cDNA prior to biotin labeling. cRNA was synthesized by in vitro transcription of cDNA and simultaneously labeled with biotin-NTP mix. The samples were filtered to recover biotinilated cRNA. Assessment of cRNA concentration, purity and quality was done using the Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). All the samples presented high quality and purity (260/280 ratio of 2.0).
Hybridization protocol
Labeled cRNA targets were fragmented at 94ºC for 20 minutes prior hybridization with arrays at 37ºC during 18 hours. After the hybridization, the bioarrays were washed and bound targets were detected following incubation with Cy5-Streptavidin (GE Healthcare, UK). The bioarrays were washed, dried and protected from light. All samples processed in parallel and the arrays were incubated simultaneously using the same working solution to limit technical variation across the experiments.
Scan protocol
Following the hybridization, bioarrays were scanned immediately in a GenePix 4000B scanner (Molecular Dynamics, USA).
Description
WKY_Nicotine_Pool1
Data processing
CodeLink Expression Analysis software (GE Healthcare) was used to extract background-subtracted spot intensities from microarray images.
To make experiments comparable, intensity data from different hybridizations were normalized by the quantile method.
