Wta system

For each sample, RNA was extracted using the QIAGEN Micro RNeasy kit Plus. cDNA was synthesized and amplified using the Ovation Pico WTA System, labeled using the Encore Biotin module and loaded on an Affymetrix GeneChip® HT HG-U133⁺ PM Array Plate which is designed to assess expression levels of transcripts in the human genome. Transcriptome profiling was performed using two successive batches of cDNA synthesized from each cohort.
Differentially expressed transcripts (DETs) detected by microarray were assessed by qPCR as previously described^{19 (link),21 (link)} using the cDNA template obtained from cohort 1 samples. Transcripts selected for validation had a differential expression between subject groups >20% and a high level of expression (Robust Multi-array Average (RMA) normalized value >7). Primer sets were designed outside of the Affymetrix target except when precluded by the small size of some transcripts (Supplemental Table 2). Selected DETs were also assessed by qPCR in grey matter homogenates of DLPFC area 9; for these samples, total RNA was converted to cDNA using the High Capacity cDNA Archive Kit from Applied Biosystems (Foster City, CA). Primer set efficiencies and normalizers for qPCR are described in Supplemental Methods and Supplemental Table 2.