Strain: C57BL/6J Gender: F Age: 8 weeks Treatment: 1 hour following nicotine-induced seizures
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from whole brains using TRI REAGENT (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions, and further purified on a Qiagen RNeasy mini kit (Qiagen Sciences, Germantown, MD)
Label
biotin-labeled
Label protocol
10 mg of total RNA were used to synthesize double-stranded cDNA with the Superscript Choice System (Invitrogen, Carlsbad, CA) and 100 pmole of oligo-dT primer attached to a T7 promoter sequence (Genset, Paris, France). The double-stranded cDNAs were phenol/chloroform extracted with Phase Lock Gels tubes (Eppendorf, Hamburg, Germany). In vitro transcription (IVT) of the double-stranded cDNA products was performed with an Enzo BioArray High Yield Transcript labeling kit in the presence of biotin-labeled nucleotides (Affymetrix, Inc., Santa Clara, CA). Following purification by an RNeasy mini kit (Qiagen Sciences), the biotin-labeled cRNA products were subjected to alkaline lysis (200mM Tris-acetate pH 8.1, 500mM potassium acetate, 150mM magnesium acetate).
Hybridization protocol
Fragmented cRNAs were hybridized overnight at 45°C to Affymetrix Gene Chip Murine Genome U74Avr2 arrays (Affymetrix, Inc.).
Scan protocol
Affymetrix Gene-Array Scanner
Description
Total RNA was extracted from whole brain. Total RNA isolation, target preprocessing and hybridization to the U74Av2 microarray were conducted according to Affymetrix’s instructions.
Data processing
The signal values were generated by statistical algorithm implanted in Affymetrix Suite Version 5.0 software (MAS5, Affymetrix, Inc.) The detection calls were calculated, based on the detection p-value, as following: probe sets with p-value of more than 0.06 were designated as absent, p-value of more than 0.04 and less than 0.06 as marginal and p-value of less than 0.04 as present. For interarray comparisons, data from each array was scaled using MAS5 software, and the mean intensity for each array was adjusted by a scaling factor to a set target intensity of 150.
