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Selection of reference gene expression in a schizophrenia brain cohort.

ABSTRACT: In order to conduct postmortem human brain research into the neuropatho-logical basis of schizophrenia, 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 with schizophrenia had significantly less PPIA and 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. When schizophrenia cases are adequately matched to control cases subtle differences in gene expression can be reliably detected.

SUBMITTER: Weickert CS

PROVIDER: S-EPMC2950262 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Selection of reference gene expression in a schizophrenia brain cohort.

Weickert Cynthia Shannon CS Sheedy Donna D Rothmond Debora A DA Dedova Irina I Fung Samantha S Garrick Therese T Wong Jenny J Harding Antony J AJ Sivagnanansundaram Sinthuja S Hunt Clare C Duncan Carlotta C Sundqvist Nina N Tsai Shan-Yuan SY Anand Jasna J Draganic Daren D Harper Clive C

The Australian and New Zealand journal of psychiatry 20100101 1

<h4>Objective</h4>In order to conduct postmortem human brain research into the neuropatho-logical basis of schizophrenia, 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.<h4>Met ...[more]

PMID: 20073568

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Project description:Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods.We recently demonstrated that relative gene expression from qRT-PCR data normalized using GAPDH, ALG9 and RPL13A expression values mirrored relative expression using quantile normalization in Robust Multichip Analysis (RMA) on the Affymetrix GeneChip rhesus Macaque Genome Array.Having shown that qRT-PCR and Affymetrix GeneChip data from the same hormone replacement therapy (HRT) study yielded concordant results, we used quantile-normalized gene microarray data to identify the most stably expressed among probe sets for prospective internal reference genes across three brain regions from the HRT study and an additional study of normally menstruating rhesus macaques (cycle study). Gene selection was limited to 575 previously published human "housekeeping" genes. Twelve animals were used per study, and three brain regions were analyzed from each animal. Gene expression stabilities were determined using geNorm, NormFinder and BestKeeper software packages.Sequences co-annotated for ribosomal protein S27a (RPS27A), and ubiquitin were among the most stably expressed under all conditions and selection criteria used for both studies. Higher annotation quality on the human GeneChip facilitated more targeted analysis than could be accomplished using the rhesus GeneChip. In the cycle study, multiple probe sets annotated for actin, gamma 1 (ACTG1) showed high signal intensity and were among the most stably expressed.Using gene microarray analysis, we identified genes showing high expression stability under various sex-steroid environments in different regions of the rhesus macaque brain. Use of quantile-normalized microarray gene expression values represents an improvement over traditional methods of selecting internal reference genes for PCR analysis.

Project description:BACKGROUND: Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L). RESULTS: Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes. CONCLUSIONS: The use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.

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Selection of reference gene expression in a schizophrenia brain cohort.

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Selection of reference gene expression in a schizophrenia brain cohort.

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