Total RNA was extracted from frozen BA10 using a Polytron type homogenizer (YellowLine DI 25 Basic) and TriZol reagent (Invitrogen, Paisley, UK) in a ratio of 1 ml of TriZol to 20 mg of tissue. RNA was further purified using RNeasy mini-columns (Qiagen, Valencia, CA, USA) including on-column DNAse-1 step and elution in water. Although pH was analysed in brain lysates using a pH meter, this was not considered to be rigorous enough to exclude or include samples and instead the RNA integrity number (RIN) was used to assess the quality of the RNA as the primary inclusion criterion. The quantity of extracted RNA was determined by spectrophotometry and quality was assessed using an Agilent 2100 Bioanalyzer (South Plainfield, NJ, USA) to determine the RIN. Based on the RIN, samples were classified into three quality groups—pass (RIN >7.0); borderline (RIN 6.0–7.0); fail (RIN<6.0). Following classification, there were 41 pass (RIN range of samples 7.0–9.0: average=7.7), 16 borderline (RIN range of samples 6.0–6.9; average=6.4) and 5 fail samples. Samples in the fail category were excluded from the study, and the remaining samples were randomized into four batches, containing an equal number of schizophrenic/control and male/female samples, for target generation and hybridization.
Label
biotin
Label protocol
For each batch, 10ug total RNA was processed to Biotin-labeled cRNA and hybridized to HG-U133_Plus_2.0 GeneChips in accordance with the Affymetrix protocol (Affymetrix, Santa Clara, CA, USA,).
Hybridization protocol
For each batch, 10ug total RNA was processed to Biotin-labeled cRNA and hybridized to HG-U133_Plus_2.0 GeneChips in accordance with the Affymetrix protocol (Affymetrix, Santa Clara, CA, USA,).
Scan protocol
Arrays were scanned on a GeneChip Scanner 3000 and fluorescence intensity for each feature of the array was obtained by using GeneChip Operating Software (Affymetrix).
Description
none
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 150.
