Validating reference genes using minimally transformed qpcr data: findings in human cortex and outcomes in schizophrenia

BACKGROUND: It is common practice, when using quantitative real time polymerase chain reaction (qPCR), to normalise levels of mRNA to reference gene mRNA which, by definition, should not vary between tissue, with any disease aetiology or after drug treatments. The complexity of human CNS means it unlikely that any gene could fulfil these criteria. METHODS: To address this issue we measured levels of mRNA for six potential reference genes (GAPDH, PPIA, SNCA, NOL9, TFB1M and SKP1) in three cortical regions (Brodmann's areas (BA) 8, 9 and 44) from 30 subjects with schizophrenia and 30 age and sex matched controls. We used a structured statistical approach to examine the characteristics of these data to determine their suitability as reference genes. We also analysed our data using reference genes selected by rank as defined using the average of the standard deviation of pair-gene ΔCt and the BestKeeper, NormFinder and geNorm algorithms to determine if they suggested the same reference genes. RESULTS: Our minimally derived data showed that levels of mRNA for all of the six genes varied between cortical regions and therefore no gene fulfilled the absolute requirements for use as reference genes. As levels of some mRNA for some genes did not vary with diagnoses within a cortical region from subjects with schizophrenia compared to controls, we normalised levels of mRNA for all the other genes to mRNA for one, two or three reference genes in each cortical region. This showed that using the geometric mean of at least two reference genes gave more reproducible results. Finally, using the reference gene ranking protocols the average of the standard deviation of pair-gene ΔCt, BestKeeper, NormFinder and geNorm we showed that these approaches ranked potential reference genes differently. We then showed that outcomes of comparing data from subjects with schizophrenia and controls varied depending on the reference genes chosen. CONCLUSIONS: Our data shows that the selection of reference genes is a significant component of qPCR study design and therefore the process by which reference genes are selected must be clearly listed as a potential confound in studying gene expression in human CNS. This should include showing that, using minimally derived qPCR data, levels of mRNA for proposed reference genes does not vary with variables such as diagnoses and CNS region

Decreased kainate receptors in the hippocampus of apolipoprotein D knockout mice

Producción CientíficaApolipoprotein D (ApoD) has many actions critical to maintaining mammalian CNS function. It is therefore significant that levels of ApoD have been shown to be altered in the CNS of subjects with schizophrenia, suggesting a role for ApoD in the pathophysiology of the disorder. There is also a large body of evidence that cortical and hippocampal glutamatergic, serotonergic and cholinergic systems are affected by the pathophysiology of schizophrenia. Thus, we decided to use in vitro radioligand binding and autoradiography tomeasure levels of ionotropic glutamate, somemuscarinic and serotonin 2Areceptors in theCNS ofApoD-/- and isogenic wild-type mice. These studies revealed a 20% decrease(mean±SEM: 104±10.2 vs. 130±10.4 fmol/mg ETE) in the density of kainate receptors in the CA 2–3 of the ApoD-/- mice. In addition therewas a global decrease inAMPA receptors (F1,214=4.67, pb0.05) and a global increase in muscarinic M2/M4 receptors (F1,208=22.77, pb0.0001) in the ApoD-/- mice that did not reach significance in any single cytoarchitectural region. We conclude that glutamatergic pathways seem to be particularly affected in ApoD-/- mice and this may contribute to the changes in learning and memory, motor tasks and orientation-based tasks observed in these animals, all of which involve glutamatergic neurotransmission

Non-Coding RNA as Novel Players in the Pathophysiology of Schizophrenia

Schizophrenia is associated with diverse changes in the brain’s transcriptome and proteome. Underlying these changes is the complex dysregulation of gene expression and protein production that varies both spatially across brain regions and temporally with the progression of the illness. The growing body of literature showing changes in non-coding RNA in individuals with schizophrenia offers new insights into the mechanisms causing this dysregulation. A large number of studies have reported that the expression of microRNA (miRNA) is altered in the brains of individuals with schizophrenia. This evidence is complemented by findings that single nucleotide polymorphisms (SNPs) in miRNA host gene sequences can confer an increased risk of developing the disorder. Additionally, recent evidence suggests the expression of other non-coding RNAs, such as small nucleolar RNA and long non-coding RNA, may also be affected in schizophrenia. Understanding how these changes in non-coding RNAs contribute to the development and progression of schizophrenia offers potential avenues for the better treatment and diagnosis of the disorder. This review will focus on the evidence supporting the involvement of non-coding RNA in schizophrenia and its therapeutic potential

Additional file 2: Figure S1. of Validating reference genes using minimally transformed qpcr data: findings in human cortex and outcomes in schizophrenia

Examples of plate wide melt curves for GAPDH, PPIA, SNCA, NOL 9, TFB1M and SKP 1. (DOCX 3905 kb

Additional file 1: Table S1. of Validating reference genes using minimally transformed qpcr data: findings in human cortex and outcomes in schizophrenia

Primer sequences and accession numbers for the primers used in qPCR. (DOCX 15 kb

Additional file 4: Table S3. of Validating reference genes using minimally transformed qpcr data: findings in human cortex and outcomes in schizophrenia

The relationships between levels of cortical mRNA and donor age, post-mortem interval (PMI), CNS pH, duration of illness (DI) and RNA integrity number (RIN). Relationships where the regression line deviated significantly from a slope of zero are bolded and in italics. (DOCX 24 kb

Studies on Prostaglandin-Endoperoxide Synthase 1: Lower Levels in Schizophrenia and After Treatment with Antipsychotic Drugs in Conjunction with Aspirin.

BACKGROUND: Antipsychotic drugs plus aspirin (acetylsalicylic acid), which targets prostaglandin-endoperoxide synthase 1 (PTGS1: COX1), improved therapeutic outcomes when treating schizophrenia. Our microarray data showed higher levels of PTGS1 mRNA in the dorsolateral prefrontal cortex from subjects with schizophrenia of long duration of illness, suggesting aspirin plus antipsychotic drugs could have therapeutic effects by lowering PTGS1 expression in the cortex of subjects with the disorder. METHODS: We used Western blotting to measure levels of PTSG1 protein in human postmortem CNS, rat and mouse cortex, and cells in culture. RESULTS: Compared with controls, PTGS1 levels were 41% lower in the dorsolateral prefrontal cortex (P<.01), but not the anterior cingulate or frontal pole, from subjects with schizophrenia. Levels of PTGS1 were not changed in the dorsolateral prefrontal cortex in mood disorders or in the cortex of rats treated with antipsychotic drugs. There was a strong trend (P=.05) to lower cortical PTGS1 10 months after mice were treated postnatally with polyinosinic-polycytidylic acid sodium salt (Poly I:C), consistent with cortical PTGS1 being lower in adult mice after exposure to an immune activator postnatally. In CCF-STTG1 cells, a human-derived astrocytic cell line, aspirin caused a dose-dependent decrease in PTGS1 that was decreased further with the addition of risperidone. CONCLUSIONS: Our data suggest low levels of dorsolateral prefrontal cortex PTGS1 could be associated with the pathophysiology of schizophrenia, and improved therapeutic outcome from treating schizophrenia with antipsychotic drugs augmented with aspirin may be because such treatment lowers cortical PTGS1