Genechip wt terminal labeling kit

The Ambion WT Expression Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to generate a cDNA strand that was fragmented using the GeneChip WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA). The obtained fragments were then hybridized onto an Affymetrix Human Gene 2.1 ST Array Strip. Subsequent fluidization and scanning steps were performed with the Affymetrix GeneAtlas System, with designated software (Affymetrix, Santa Clara, CA, USA). Bioinformatics analysis was performed in the statistical programming language R using BioConductor software. The robust multiarray average (RMA) normalization algorithm in the “Affy” library was used for the normalization, background correction, and calculation of the expression level of all tested genes. A detailed description of the mRNA microarray was described in our previous articles [14 (link),26 (link)].

After the tissue was homogenized, total RNA was extracted from the hippocampus, amygdala, thalamus and prefrontal cortex of each subject using the TRIzol reagent (Invitrogen Life Technologies, Germany) as previously described^{18 (link)}. Genomic DNA was removed using the RNeasy Plus kit (Qiagen, Germany). Prior to sample preprocessing, RNA integrity was assessed with the RNA 6000 nano kit using the Bioanalyzer 2100 instrument (Agilent, Germany). RNA integrity numbers ranged between 6.5 and 8.2. 200 ng of each sample were processed with the whole transcript (WT) expression kit (Ambion, Germany), i.e., subjected to RNA amplification via reverse transcription to double-stranded cDNA and subsequent in vitro transcription; this was followed by another round of reverse transcription yielding single-stranded DNA in sense orientation. Hybridisation cocktails were produced after fragmentation and biotin labeling of target DNAs following the protocol of the GeneChip WT terminal labeling kit (Affymetrix, Canada). Microarray hybridisation to GeneChip Human Transcriptome Array 2.0 (Affymetrix, Canada) was performed individually according to the manufacturer's protocol using the Fluidics Station 450 with the program FS450_0007. CEL files from scanned microarrays were produced with the expression console (Affymetrix, Canada). The hybridization experiment was repeated twice^{19 (link)}.