DOI: 10.6084/m9.figshare.20520378
title: qPCR: Neurotransmission-related gene expression in post-mortem tissue from frontal cortex region Brodmann Area 10 (BA10) from subjects with Schizophrenia and Bipolar Disorder

description: <strong>DATA ABSTRACT:</strong>
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Brodmann Area 10 (BA10) is the largest cytoarchitectonic region of the human cortex, performing complex integrative functions. BA10 undergoes intensive adolescent grey matter pruning around the average age of onset for Bipolar disorder (BP) and Schizophrenia (SCHIZ), and its dysfunction is likely to underly aspects of their shared symptomology. In this study, we investigated the role of BA10 neurotransmission-related gene expression in BP and SCHIZ. We performed qPCR to measure the expression of 115 neurotransmission-related targets in control, BP, and SCHIZ post-mortem samples (<em>n</em>=72). We chose this method for its high sensitivity to detect low-level expression. To improve interpretation, we compiled an unusually large database of clinical metadata for our samples. We used this data to explore the relationship between BA10 gene expression, therapeutics, substances of abuse, and symptom profiles, and validated these findings with publicly-available datasets. 
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<strong>DATA FILES:</strong>
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<strong>1)"TableS1_AllSampleDemographics_wExploratory_ForFigShare.xlsx"</strong>: Contains the metadata for the samples for all subjects ("Subject ID"), including demographics, clinical characteristics, pre- and post-mortem factors, and technical information.
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<strong>2)"Concatenated_GabaGlu_ForFigShare.csv" and "Concatenated_DA5HT_ForFigShare.csv": </strong>The qPCR output for the experiment focused on targets related to the GABA and glutamate ("GabaGlu") neurotransmitter systems and Dopamine and Serotonin ("DA5HT") neurotransmitter systems. The samples for all subjects ("Subject ID") were run in either duplicate or quadruplicate.
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<strong>RELEVANT METHODS:</strong>
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This research was overseen and approved by the University of Michigan Institutional Review Board, Pritzker Neuropsychiatric Disorders Research Consortium, and the University of California Irvine Institutional Review Board. Key Resources (<strong>Table S1</strong>) and full methodological details are documented in the supplement. 
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Human samples were collected through the University of California-Irvine Pritzker Brain Donor Program with informed consent from next of kin (n=72, CTRL: <em>n</em>=27, BPD: <em>n</em>=21, SCHIZ: <em>n</em>=24; <strong>Table S2, Fig S1</strong>). A detailed psychological autopsy was performed using coroner records, medical records, and interviews with next-of-kin (<strong>Appendix 1).</strong> This information was used to confirm BP and SCHIZ diagnosis, and ensure absence of neurological or psychiatric disorder in CTRL subjects or their first-degree relatives. Other clinical information was summarized as 49 exploratory variables (<strong>Fig S2</strong>) denoting the presence or absence of 1) medication, 2) exposure to alcohol or drugs of abuse, and 3) diagnosis-related symptoms. 
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Brains were extracted during autopsy and kept on ice until being sliced into 1 cm thick coronal slabs, then snap-frozen for storage (-80℃) until microdissection. After counterbalancing processing batches by diagnosis, samples were blinded, and the foremost rostral slab from the left hemisphere was sub-dissected to obtain blocks averaging 500 µg containing lateral BA10 (<strong>Fig S3</strong>), a subregion implicated in SCHIZ. RNA was extracted using TRIzol™ and purified (RNeasy® Mini Kit). cDNA was synthesized (iScript Reverse Transcription Supermix kit) and analyzed in duplicate via qPCR (Applied Biosystems ViiA 7 real time PCR system) using two sets of ThermoFisher Scientific Taqman Gene Expression Array qPCR cards: 1) “Human GABA Glutamate” (REF#4342259): 84 targets (12 reference genes), 2) “Dopamine Serotonin” (REF#4342253): 31 targets (17 reference genes) (<strong>Table S3</strong>). These cards were preloaded with a complete list of targets for the main neurotransmitter systems in the frontal cortex: glutamate, GABA, dopamine, and serotonin, including receptors, transporters, metabolic enzymes, and other associated molecules. The cards were further customized to include several well-known markers for interneuron subtypes (SST, PVALB, CALB1) and astrocytes (AQP4, GJA1, GFAP, KCNJ10, S100B) to enhance the interpretation of neurotransmission-related data. Prior to differential expression analysis, the qPCR quantification cycle (Cq) data for targets was normalized using the average reference gene expression for each sample to produce -deltaCq values.
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