2.1. Exon Array (Experiment 1)
Anterior cingulate cortex (ACC) samples were used in an exon array (Affymetrix HuEx 1.0 ST, Santa Clara, CA, USA. ACC samples from 9 bipolar disorder patients and 11 healthy controls were used in this study for initial microarray analysis. The demographics are shown in Table 1. Approximately 100 mg of dissected frozen tissue was homogenized using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) following the standard RNA Trizol isolation procedure: 1 mL Trizol was added to frozen brain, then Trizol mixture was homogenized for 30 s twice at 7500 rpm using Tissue Tearor (Biospec Products, Inc., Bartlesville, OK, USA) in ice. The mixture was subsequently incubated at room temperature for 5 min, 200 µL of chloroform added, the tube shaken by hand for 30 s and the mixture then incubated for 2–3 min at reverse transcription (RT), before being centrifuged at 12,000 g for 15 min at 4 °C with the Eppendorf Centrifuge 5417R (Eppendorf, Hauppauge, NY, USA). The supernatant containing the upper aqueous phase was transferred to a new tube, mixed with 500 µL of isopropyl alcohol and incubated for 15 min at RT and centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was removed, and the pellet was washed with 1 mL of iced 75% ethanol, by brief vortex, then centrifuged at 7500 g for 10 min at 4 °C. Ethanol was decanted, and RNA pellet was dried at RT for 5–10 min in a laboratory hood by opening tube lid; RNA was then dissolved in 50 µL DEPC-treated water by gently mixing on ice. RNA was stored in a −80 °C freezer. The resulting total RNA was cleaned of low molecular weight fragments by passing through a Qiagen column, and checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) for quality control using RNA integrity number. The concentrations measured on a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) were adjusted to 1 µg/µL.
GeneChip Whole Transcript Sense Assay Protocol:
The Affymetrix Human GeneChip Exon 1.0 ST arrays were run following the manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA). Briefly, 2 μg of purified total RNA underwent ribosomal RNA removal using the RiboMinus Human/Mouse Transcriptome Isolation Kit (Invitrogen). The reduced RNA was then reverse transcribed to cDNA using random hexamers tagged with a T7 promoter sequence followed by a second strand cDNA synthesis using DNA polymerase (GeneChip WT cDNA Synthesis and Amplification Kit, Affymetrix). The resulting double-stranded cDNA was then used for amplification of antisense cRNA using the Gene Chip Sample Cleanup Module (Affymetrix). A second cycle cDNA synthesis was performed using random primers to reverse transcribe the cRNA into sense single-stranded DNA. The DNA was then enzymatically fragmented and labeled using the GeneChip WT Terminal Labeling Kit (Affymetrix). The hybridization cocktail consisting of the labeled sample, Control Oligonucleotide B2, and 20× Eukaryotic Hybridization Controls were heated for 5 min at 99 °C and cooled for 5 min at 45 °C, then centrifuged 1 min. A volume of 200 µL was loaded onto the Affymetrix Human Gene Chip Exon 1.0 ST Arrays and the arrays were placed in a 45 °C hybridization oven, at 60 rpm, to incubate for 17 h. The GeneChip Hybridization, Wash and Stain Kit (Affymetrix) was used with the Fluidics Station 450_0001 protocol. Arrays were then scanned on the GeneChip Scanner 3000 7G (Affymetrix). All exon array samples were processed in the same batch by one person.
Each CEL file from the Affymetrix HuEx 1.0 ST was imported into Partek Genomics Suite 6.6 using background subtraction and elimination of probes with common SNPs from analysis of exon array data following the method of Gamazon et al. 2010 [30]. The initial count of probesets on the array is 1.1 million (see Supplementary Methods), elimination of common SNPs in probes reduced the probeset count by ~350,000 probesets. Each CEL file was analyzed together using robust multiarray analysis (RMA) [31]. Resulting expression values of probes were averaged in each probeset. Each probeset was aligned to a unique RefSeq gene, and we report only findings that are associated with full-length mRNA and have coverage by at least two probesets. This reduced the total probesets analysis to 230,659 probesets representing 11,807 full-length RefSeq genes. The diagnosis by probeset interaction was calculated in Partek (Supplementary Methods), and the interaction p-values cut-off were determined after Bonferroni correction.