Genome-wide expression assay comparison across frozen and fixed postmortem brain tissue samples (original) (raw)
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Methodological considerations for gene expression profiling of human brain
Journal of Neuroscience Methods, 2007
Gene expression profiles of postmortem brain tissue represent important resources for understanding neuropsychiatric illnesses. The impact(s) of quality covariables on the analysis and results of gene expression studies are important questions. This paper addressed critical variables which might affect gene expression in two brain regions. Four broad groups of quality indicators in gene expression profiling studies (clinical, tissue, RNA, and microarray quality) were identified. These quality control indicators were significantly correlated, however one quality variable did not account for the total variance in microarray gene expression. The data showed that agonal factors and low pH correlated with decreased integrity of extracted RNA in two brain regions. These three parameters also modulated the significance of alterations in mitochondrial-related genes. The average F-ratio summaries across all transcripts showed that RNA degradation from the AffyRNAdeg program accounted for higher variation than all other quality factors. Taken together, these findings confirmed prior studies, which indicated that quality parameters including RNA integrity, agonal factors, and pH are related to differences in gene expression profiles in postmortem brain. Individual candidate genes can be evaluated with these quality parameters in post hoc analysis to help strengthen the relevance to psychiatric disorders. We find that clinical, tissue, RNA, and microarray quality are all useful variables for collection and consideration in study design, analysis, and interpretation of gene expression results in human postmortem studies.
A cross-laboratory comparison of expression profiling data from normal human postmortem brain
Neuroscience, 2010
Expression profiling of post-mortem human brain tissue has been widely used to study molecular changes associated with neuropsychiatric diseases as well as normal processes such as aging. Changes in expression associated with factors such as age, gender or postmortem interval are often more pronounced than changes associated with disease. Therefore in addition to being of interest in their own right, careful consideration of these effects are important in the interpretation of disease studies. We performed a large meta-analysis of genome-wide expression studies of normal human cortex to more fully catalogue the effects of age, gender, postmortem interval and brain pH, yielding a "metasignature" of gene expression changes for each factor. We validated our results by showing a significant overlap with independent gene lists extracted from the literature. Importantly, meta-analysis identifies genes which are not significant in any individual study. Finally, we show that many schizophrenia candidate genes appear in the meta-signatures, reinforcing the idea that studies must be carefully controlled for interactions between these factors and disease. In addition to the inherent value of the meta-signatures, our results provide critical information for future studies of disease effects in the human brain.
Biological Psychiatry, 2004
There are major concerns that specific agonal conditions, including coma and hypoxia, might affect ribonucleic acid (RNA) integrity in postmortem brain studies. We report that agonal factors significantly affect RNA integrity and have a major impact on gene expression profiles in microarrays. In contrast to agonal factors, gender, age, and postmortem factors have less effect on gene expression profiles. The Average Correlation Index is proposed as a method for evaluating RNA integrity on the basis of similarity of microarray profiles. Reducing the variance due to agonal factors is critical in investigating small but validated gene expression differences in messenger RNA levels between psychiatric patients and control subjects.
Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue
Journal of Visualized Experiments, 2009
We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons. Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51μg, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.
Application and optimization of microarray technologies for human postmortem brain studies
Biological Psychiatry, 2004
A number of microarray investigations using human postmortem brain tissue have been published recently, exploring a multitude of human brain disorders with the aim of unraveling the underlying pathologies. Although the technology is still developing and lacks sufficient sensitivity with regard to detecting splice variants and low abundance transcripts, microarrays are becoming the prominent method for candidate gene screening in complex neuropsychiatric disorders. The use of postmortem tissue harbors a variety of potential pitfalls, however, which could result in unreliable or, at worst, meaningless results. During the course of our large-scale gene expression study on 150 human postmortem brain samples, using more than 200 Affymetrix GeneChips, we have identified several aspects within microarray experimental procedure that allows for the early identification of potentially unreliable samples. The general application of the guidelines and technical tips described here increase the efficiency, reliability, and amount of data generated by this powerful screening technology while reducing superfluous consumption of time and resources.
Postmortem interval effect on RNA and gene expression in human brain tissue
Cell and Tissue Banking
Banked tissue is essential to the study of neurological disease but using postmortem tissue introduces a number of possible confounds. Foremost amongst these are factors relating to variation in postmortem interval (PMI). Currently there are conflicting reports on how PMI affects overall RNA integrity, and very few reports of how gene expression is affected by PMI. We analyzed total RNA extracted from frozen cerebellar cortex from 79 deceased human subjects enrolled in the Banner Sun Health Research Institute Brain and Body Donation Program. The PMI, which ranged from 1.5 to 45 hours, correlated with overall RNA quality measures including RNA Integrity Number (RIN) (r = − 0.34, p = 0.002) and RNA quantitative yield (r = − 0.25, p = 0.02). Additionally, we determined the expression of 89 genes using a PCR-based gene expression array (RT 2 Profiler TM PCR Array: Human Alzheimer's Disease; SABiosciences TM , Frederick, MD). A greater proportion of genes had decreased rather than increased expression with increasing PMI (65/89 vs 20/89; p < 0.0001). Of these, transcripts from the genes ADAM9, LPL, PRKCG, and SERPINA3 had significantly decreased expression with increasing PMI (p < 0.01). No individual gene transcripts had significantly increased expression with increasing PMI. In conclusion, it is apparent that RNA degrades progressively with increasing PMI and that measurement of gene expression in brain tissue with longer PMI may give artificially low values. For tissue derived from autopsy, a short PMI optimizes its utility for molecular research.
Frontiers in Molecular Neuroscience, 2016
Different cell types and multiple cellular connections characterize the human brain. Gene expression analysis using a specific population of cells is more accurate than conducting analysis of the whole tissue homogenate, particularly in the context of neurodegenerative diseases, where a specific subset of cells is affected by the different pathology. Due to the difficulty of obtaining homogenous cell populations, gene expression in specific cell-types (neurons, astrocytes, etc.) has been understudied. To leverage the use of archive resources of frozen human brains in studies of neurodegenerative diseases, we developed and calibrated a method to quantify cell-type specific-neuronal, astrocytes-expression profiles of genes implicated in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Archive human frozen brain tissues were used to prepare slides for rapid immunostaining using cell-specific antibodies. The immunoreactive-cells were isolated by Laser Capture Microdissection (LCM). The enrichment for a particular cell-type of interest was validated in post-analysis stage by the expression of cell-specific markers. We optimized the technique to preserve the RNA integrity, so that the RNA was suitable for downstream expression analyses. Following RNA extraction, the expression levels were determined digitally using nCounter Single Cell Gene Expression assay (NanoString Technologies ®). The results demonstrated that using our optimized technique we successfully isolated single neurons and astrocytes from human frozen brain tissues and obtained RNA of a good quality that was suitable for mRNA expression analysis. We present here new advancements compared to previous reported methods, which improve the method's feasibility and its applicability for a variety of downstream molecular analyses. Our new developed method can be implemented in genetic and functional genomic research of neurodegenerative diseases and has the potential to significantly advance the field.
Critical Factors in Gene Expression in Postmortem Human Brain: Focus on Studies in Schizophrenia
Biological Psychiatry, 2006
Background: Studies of postmortem human brain are important for investigating underlying pathogenic molecular mechanisms of neuropsychiatric disorders. They are, however, confounded by pre-and postmortem factors. The purpose of this study was to identify sources of variation that will enable a better design of gene expression studies and higher reliability of gene expression data. Methods: We assessed the contribution of multiple variables to messenger RNA (mRNA) expression of reference (housekeeping) genes measured by reverse transcriptase-polymerase chain reaction (RT-PCR) by multiple regression analysis in a large number (N ϭ 143) of autopsy samples from the hippocampus and white and grey matter of the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia and normal control subjects. Results: The strongest predictor of gene expression was total RNA quality. Other significant factors included pH, postmortem interval, age and the duration of the agonal state, but the importance of these factors depended on transcript measured, brain region analyzed, and diagnosis. The quality of RNA obtained from the DLPFC white matter was also adversely affected by smoking. Conclusions: Our results show that normalization of expression data of target genes with a geometric mean of multiple housekeeping genes should be used to control for differences in RNA quality between samples. The results also suggest that accurate assessment of other confounding factors and their inclusion as regressors in the analysis is critical for obtaining reliable and accurate quantification of mRNA expression.