Elsevier

Biological Psychiatry

Volume 60, Issue 6, 15 September 2006, Pages 650-658
Biological Psychiatry

Original article
Critical Factors in Gene Expression in Postmortem Human Brain: Focus on Studies in Schizophrenia

https://doi.org/10.1016/j.biopsych.2006.06.019Get rights and content

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 = 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.

Section snippets

Subjects

Human brain specimens were collected in the Section on Neuropathology of the Clinical Brain Disorders Branch at the National Institute of Mental Health (NIMH) through the Offices of the Chief Medical Examiner of the District of Columbia and of Northern Virginia, after autopsy, and through tissue donations via funeral homes. Informed consent to study brain tissue was obtained from the surviving next-of-kin for all cases, according to Protocol #90-M-0142 approved by the NIMH/National Institutes

Subject Selection

Fifty-three subjects with a documented history of schizophrenia and 90 control subjects were identified as suitable for further studies after excluding individuals having: 1) a primary psychiatric diagnosis other than schizophrenia, such as a mood disorder, substance abuse, or anxiety disorder; 2) clinical history and/or neuropathological abnormalities consistent with a primary neurological disorder such as Alzheimer’s disease, Parkinson’s disease, or diffuse Lewy Body disease; 3) a positive

Discussion

In this study, we report the results of a series of analyses aimed at parsing variance in gene expression in postmortem brain tissue. We also estimated a fold of between-group differences in gene expression that could be resolved with these brain tissue samples. We confirmed the often-reported observations that RNA quality, pH, PMI, and age result in changes in gene expression and that patients with schizophrenia as a sample population tend to have more extreme values in these confounders than

Summary

We have identified and/or validated factors that should be considered when assembling a cohort of postmortem brains for the investigation of gene expression. We found that the most important factor affecting expression levels of housekeeping genes, regardless of the diagnosis or brain tissue type, was total RNA quality. Accurate and reliable assessment of RNA quality should thus be an essential step in gene expression studies. Other factors to be considered include pH, PMI, age, agonal state,

References (53)

  • S. Leonard et al.

    Smoking and mental illness

    Pharmacol Biochem Behav

    (2001)
  • D.A. Lewis

    The human brain revisited: Opportunities and challenges in postmortem studies of psychiatric disorders

    Neuropsychopharmacology

    (2002)
  • J. Lupberger et al.

    Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR

    Mol Cell Probes

    (2002)
  • M.K. Park et al.

    Cerebral white matter lesions and hypertension status in the elderly Korean: The Ansan Study

    Arch Gerontol Geriatr

    (2005)
  • P. Preece et al.

    Quantifying mRNA in postmortem human brain: Influence of gender, age at death, postmortem interval, brain pH, agonal state and inter-lobe mRNA variance

    Brain Res Mol Brain Res

    (2003)
  • P. Preece et al.

    An optimistic view for quantifying mRNA in post-mortem human brain

    Brain Res Mol Brain Res

    (2003)
  • R. Ravid et al.

    Brain banking and the human hypothalamus—factors to match for, pitfalls and potentials

    Prog Brain Res

    (1992)
  • M. Ricicova et al.

    Comparative analyses of Saccharomyces cerevisiae RNAs using Agilent RNA 6000 Nano Assay and agarose gel electrophoresis

    FEMS Yeast Res

    (2003)
  • H. Tomita et al.

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

    Biol Psychiatry

    (2004)
  • E.F. Torrey et al.

    Neurochemical markers for schizophrenia, bipolar disorder, and major depression in postmortem brains

    Biol Psychiatry

    (2005)
  • C. Tricarico et al.

    Quantitative real-time reverse transcription polymerase chain reaction: Normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies

    Anal Biochem

    (2002)
  • Diagnostic and Statistical Manual of Mental Disorders, 4th ed., Text Revision

    (2000)
  • A.J. Barton et al.

    Pre- and postmortem influences on brain RNA

    J Neurochem

    (1993)
  • N.J. Bray et al.

    Haplotypes at the dystrobrevin binding protein 1 (DTNBP1) gene locus mediate risk for schizophrenia through reduced DTNBP1 expression

    Hum Mol Genet

    (2005)
  • S.A. Bustin et al.

    Quantitative real-time RT-PCR—a perspective

    J Mol Endocrinol

    (2005)
  • F. Centorrino et al.

    Inpatient antipsychotic drug use in 1998, 1993, and 1989

    Am J Psychiatry

    (2002)
  • Cited by (0)

    View full text