Selection of Reference Gene Expression in a Schizophrenia Brain Cohort

Abstract

Objective To conduct postmortem human brain research into the neuropathological basis of schizophrenia, it is critical to establish cohorts that are well-characterised and well-matched. Our objective was to determine if specimen characteristics, including: diagnosis, age, postmortem interval (PMI), brain acidity (pH), and/or the agonal state of the subject at death related to RNA quality, and to determine the most appropriate reference gene mRNAs. Methods We selected a matched cohort of 74 cases (37 schizophrenia / schizoaffective disorder cases and 37 controls cases). Middle frontal gyrus tissue was pulverised, tissue pH was measured, RNA isolated for cDNA from each case, and RNA integrity number (RIN) measurements were assessed. Using RT-PCR, we measured nine housekeeper genes and calculated a geomean in each diagnostic group. Results We found that the RINs were very good (mean 7.3) and all nine housekeeper control genes were significantly correlated with RIN. Seven of nine housekeeper genes were also correlated with pH, and two clinical variables, agonal state and duration of illness did have an effect on some control mRNAs. No major impact of PMI or freezer time on housekeeper mRNAs was detected. Our results show that people with schizophrenia had significantly less PPIA, and SDHA and tended to have less GUSB and B2M mRNA suggesting that these control genes may not be good candidates for normalisation. Conclusions In our cohort, less than 10% variability in RIN values was detected and the diagnostic groups were well matched overall. Our cohort was adequately powered (0.80–0.90) to detect mRNA differences (25%) due to disease. Our study suggests that multiple factors should be considered in mRNA expression studies of human brain tissues. When schizophrenia cases are adequately matched to control cases subtle differences in gene expression can be reliably detected.

Postmortem Brain: An Underutilized Substrate for Studying Severe Mental Illness

“…The effects of freezer storage time on dependent measures have been examined. Storage time did not affect mRNA quality or quantity (Burnet et al, 1997;Harrison et al, 1995;Stockmeier, 1997;Weickert et al, 2010). However, extended freezer storage time affected oligo dT priming of reverse transcription, suggesting that there may be degradation of the polyA tail (Johnston et al, 1997;Leonard et al, 1993).…”

“…Agonal respirations often follow cardiogenic shock (Rea, 2005), and may lead to a prolonged period of hypoxia due to agonal breathing at the end of life. Several studies have reported diminished mRNA quality associated with prolonged agonal states (Barton et al, 1993;Durrenberger et al, 2010;Harrison et al, 1991Harrison et al, , 1995Kingsbury et al, 1995;Lipska et al, 2006a, b;Tomita et al, 2004a, b;Vawter et al, 2006;Weickert et al, 2010). While subjects known to have a prolonged agonal status are typically excluded from postmortem studies, the effects of agonal status on proteins are not as well established.…”

Density of metabotropic glutamate receptors 2 and 3 (mGluR2/3) in the dorsolateral prefrontal cortex does not differ with schizophrenia diagnosis but decreases with age

“…Bonferroni correction was used to correct for multiple testing in case of significance. Spearman's correlation was used to test for any effects of continuous descriptive variables including age at death, pH (prefrontal cortex), post mortem interval (PMI), RNA integrity (RIN), freezer storage time, brain weight, brain volume, chlorpromazine equivalent (mg) of lowest, highest, lifetime and last recorded antipsychotic dose, antidepressant treatment, daily ethanol intake, smoking quantity, age of onset and duration of illness on receptor binding (for clinical data see (Weickert et al 2010). ANCOVA analysis, controlling for age of death, was performed for obtained significances when using the the Student's t-test; levels of radioligand binding in suicidal vs naturally deceased schizophrenia/schizoaffective patients as well as patients on last recorded high (>450mg/day chlorpromazine equivalent) or low (<450mg/day chlorpromazine equivalent) dose of antipsychotics were therefore studied.…”

“…All research was approved and conducted under the guidelines of the Human Research Ethics Committee at the University of Wollongong (HE99.222) and University of New South Wales (HREC 07261). Clinical assessments, selection of cases and matched controls, assessment of tissue quality and preparation of slide-mounted coronal tissue sections (14µm) were performed by the Tissue Resource Centre and the Schizophrenia Research Institute (Weickert et al 2010). All experiments and analysis was done blind to the clinical details of each case.…”

Increased expression of astrocyte markers in schizophrenia: Association with neuroinflammation

“…Post-mortem tissue from the DLPFC was obtained from 37 individuals with schizophrenia and 37 controls (demographic summary in Table 1) within the New South Wales Tissue Resource Centre collection, which has been characterized previously (Weickert et al, 2010). We also utilized our existing database of mRNA transcript expression levels determined by RNA-Seq in DLPFC from a subset of 20 individuals with schizophrenia and 20 controls (demographic summary in Table 1) described previously (Fillman et al, 2013).…”