Effect of agonal and postmortem factors on gene expression profile: quality control in microarray analyses of postmortem human brain

Abstract

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. KeywordsAdenine; Uracil-rich elements; bipolar disorder; freezer interval; iron-responsive element; major depressive disorder; postmortem interval Microarray studies using postmortem brains have become particularly valuable in profiling gene expression patterns related to psychiatric disorders of unknown etiology (Bahn et al 2001;Bunney et al 2003;Hakak et al 2001;Mirnics et al 2001;Van Deerlin et al 2002). In studies using postmortem brain tissue, the variability of ribonucleic acid (RNA) integrity has been a major concern. There have been two major factors that affect RNA integrity in gene expression studies using postmortem brain: 1) agonal factors, defined as specific agonal conditions at the time of death and agonal duration; and 2) postmortem factors, defined as the condition of the postmortem brain tissue after death, including the delay between death and the time the tissue is frozen (postmortem interval [PMI]) and the duration the brain tissue is stored in a freezer (freezer interval [FI]). Prior studies have indicated that agonal factors markedly affect the integrity of RNA, whereas postmortem factors have relatively small effects on RNA integrity (Bahn et al 2001;Barton et al 1993;Gilmore et al 1993;Harrison et al 1995;Johnson et al 1986;Kingsbury et al 1995;Leonard et al 1993;Perrett et al 1988;Ravid et al 1992;Van Deerlin et al 2002). However, because messenger (m)RNAs have been shown to vary in their vulnerability to agonal factors (Barton et al 1993;Burke et al 1991;Harrison et al 1994Harrison et al , 1995, attention should be drawn to the limitation of previous postmortem studies in this area, in which investigators have only analyzed restricted numbers of transcripts. Expression data for thousands of genes obtained by microarray technology facilitate a comprehensive evaluation of the integrity of RNA samples.In this article, we discuss issues concerning evaluation of agonal and postmortem factors and RNA integrity in microarray study of postmortem brains. The hypothesis raised in the present study was that agonal factors adversely affect integrity of mRNAs and influence microarray expression profiles more than the postmortem factor or other biological factors, such as gender, age, or diagnosis of psychiatric disorder. The hypothesis was tested with 40 well-characte...

GABAergic Neurons Immunoreactive for Calcium Binding Proteins are Reduced in the Prefrontal Cortex in Major Depression

“…It is also known that changes in tissue pH are associated with certain pathological conditions such as ischemia or head injury (Billups and Attwell, 1996;Cadoux-Hudson et al, 1990;Smith et al, 1986). Prolonged agonal state related to hypoxia may also lower brain tissue pH (Tomita et al, 2004). This is, however, unlikely to be the case in our subjects as all of them were victims of sudden death (either by suicide, or due to accidental causes).…”

Human postmortem tissue: What quality markers matter?

“…This study assumed optimal clinical diagnosis of cases, an essential feature of the process (Deep-Soboslay et al, 2005). The answers to these questions serve to suggest additional questions for the area of postmortem tissue quality and provide support for the use of postmortem brain tissue in studies of human brain research (Lewis, 2002;Tomita et al, 2004;Torrey et al, 2000).…”

“…Tomita et al developed the Agonal Factor Score (AFS) to quantify the manner of death (Tomita et al, 2004). To calculate the AFS, several factors were rated: prolonged death (PD), coma, pyrexia, hypoxia, multiple organ failure (MOF), head injury (HI), neurotoxic substance ingestion.…”

“…Several laboratories have approached the evaluation of human postmortem brain cases with suggestions for markers of tissue quality (Lee et al, 2005;Preece and Cairns, 2003;Tomita et al, 2004). We have taken several of these newer measures of tissue quality and tested them in human postmortem tissue in a variety of situations to detect the relationship of the markers to the real quality of the postmortem tissue.…”

Quantitative Analyses of Postmortem Heat Shock Protein mRNA Profiles in the Occipital Lobes of Human Cerebral Cortices: Implications in Cause of Death

“…This is primarily due to the fact that postmortem human tissues are heterogeneous. Postmortem intervals and conditions as well as antemortem factors such as age, gender, body mass, and the physiological/pathological conditions of the agonal phase can profoundly affect expression profiles and half-lives of certain gene transcripts in postmortem tissues, obscuring the employment of molecular biomarkers to investigate the cause and process of death (Durrenberger et al, 2010;Preece and Cairns, 2003;Tomita et al, 2004). Therefore, data normalization and RNA integrity have been essential issues in the field of forensic molecular pathology (Vennemann and Koppelkamm, 2010b).…”