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Multiple Comparisons and Inappropriate Statistical Testing Lead to Spurious Sex Differences in Gene Expression

      The substantial differences in the incidence and symptoms of stress, depression, and addiction between males and females have motivated studies of sex differences in the brain’s molecular responses to environmental stimuli. In a study of three mouse brain regions from the Nestler laboratory, Walker et al. (
      • Walker D.M.
      • Zhou X.
      • Cunningham A.M.
      • Lipschultz A.P.
      • Ramakrishnan A.
      • Cates H.M.
      • et al.
      Sex-specific transcriptional changes in response to adolescent social stress in the brain’s reward circuitry.
      ) reported sex-specific transcriptional responses to cocaine and baseline sex differences. They further examined the impact of social isolation on these brain gene expression patterns. However, the transcriptome data (RNA sequencing) and analysis in this study do not support the authors’ conclusions. Here, I describe two critical errors that invalidate the statistical analyses: lack of correction for multiple comparisons and drawing positive conclusions from negative (inconclusive) statistical results. I further show how a valid analysis could be performed for these data.
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      References

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      Linked Article

      • Sex-Specific Transcriptional Changes in Response to Adolescent Social Stress in the Brain’s Reward Circuitry
        Biological PsychiatryVol. 91Issue 1
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          Sex differences in addiction have been described in humans and animal models. A key factor that influences addiction in both males and females is adolescent experience. Adolescence is associated with higher vulnerability to substance use disorders, and male rodents subjected to adolescent social isolation (SI) stress form stronger preferences for drugs of abuse in adulthood. However, little is known about how females respond to SI, and few studies have investigated the transcriptional changes induced by SI in the brain’s reward circuitry.
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      • Reply to: Multiple Comparisons and Inappropriate Statistical Testing Lead to Spurious Sex Differences in Gene Expression
        Biological PsychiatryVol. 91Issue 1
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          In our recent publication (1), we used RNA sequencing of several limbic brain regions to identify sex-specific transcriptional profiles in response to cocaine that are disrupted by adolescent experience. RNA sequencing provides a powerful approach to investigate transcription on a global scale and has been instrumental in contributing to an improved understanding of brain function in recent years. For example, our laboratories have successfully employed transcriptomic analyses to identify novel key drivers important for regulating behavioral and neural abnormalities associated with stress- and addiction-related disorders [e.g., (2–7)].
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