tissue: brain anterior cingulate cortex disease state: MDD case
1µg of total high quality total RNA was reverse transcribed into single stranded cDNA, that was then converted into double stranded cDNA and purified using the Illumina TotalPrep RNA Amplification Kit. An in vitro transcription reaction was carried out overnight in the presence of biotinylated UTP and CTP to obtain biotin-labeled cRNA from the double stranded cDNA. The cRNA was purified using the same Amplification Kit, and quality control was assessed.
standard Illumina protocol
The sections were then hybridized with [35S]-labeled riboprobes (10^6 cpm/uL) in hybridization buffer at 56 °C for 16h. The hybridization buffer contained 50% formamide, 0.75 M NaCl, 20 mM 1,4-piperazine diethane sulfonic acid, pH 6.8, 10 mM ethylenediaminetetraacetic acid (EDTA), 10% dextran sulfate, 5X Denhardt’s solution (0.2 mg/mL Ficoll, 0.2 mg/mL polyvinylpyrrolidone, 0.2 mg/mL bovine serum albumin), 50 mM dithiothreitol, 0.2% sodium dodecyl sulfate, and 100 ug/mL yeast transfer RNA.
DirecHyb Gene Expression. iScan Control Software 1.3.10+
Probeset signals were extracted with the Beadarray software following the default quality control parameters and batch-normalized. GenomeStudio Gene Expression Module v1.6+