| 1 | Aust N Z J Psychiatry 2010 Jan 44: 59-70 |
|---|---|
| PMID | 20073568 |
| Title | Selection of reference gene expression in a schizophrenia brain cohort. |
| Abstract | In order to conduct postmortem human brain research into the neuropatho-logical basis ofschizophrenia, it is critical to establish cohorts that are well-characterized and well-matched. The aim of the present study was therefore 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. A matched cohort was selected of 74 subjects (schizophrenia/schizoaffective disorder, n = 37; controls, n = 37). Middle frontal gyrus tissue was pulverized, tissue pH was measured, RNA isolated for cDNA from each case, and RNA integrity number (RIN) measurements were assessed. Using quantitative reverse transcription-polymerase chain reaction, nine housekeeper genes were measured and a geomean calculated per case in each diagnostic group. 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; 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. The results show that people withschizophreniahad significantly lessPPIAand SDHA mRNA and tended to have less GUSB and B2M mRNA, suggesting that these control genes may not be good candidates for normalization. In the present cohort <10% variability in RINs was detected and the diagnostic groups were well matched overall. The cohort was adequately powered (0.80-0.90) to detect mRNA differences (25%) due to disease. The study suggests that multiple factors should be considered in mRNA expression studies of human brain tissues. Whenschizophreniacases are adequately matched to control cases subtle differences in gene expression can be reliably detected. |
| SCZ Keywords | schizophrenia |
| 2 | BMC Psychiatry 2016 -1 16: 154 |
| PMID | 27206773 |
| Title | Validating reference genes using minimally transformed qpcr data: findings in human cortex and outcomes in schizophrenia. |
| Abstract | 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. 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 withschizophreniaand 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 ?Ct and the BestKeeper, NormFinder and geNorm algorithms to determine if they suggested the same reference genes. 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 withschizophreniacompared 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 ?Ct, BestKeeper, NormFinder and geNorm we showed that these approaches ranked potential reference genes differently. We then showed that outcomes of comparing data from subjects withschizophreniaand controls varied depending on the reference genes chosen. 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. |
| SCZ Keywords | schizophrenia |