Abstract
Single-neuron gene expression studies may be especially important for understanding nervous system structure and function because of the neuron-specific functionality and plasticity that defines functional neural circuits. Cellular dissociation is a prerequisite technical manipulation for single-cell and single cell-population studies, but the extent to which the cellular dissociation process affects neural gene expression has not been determined. This information is necessary for interpreting the results of experimental manipulations that affect neural function such as learning and memory. The goal of this research was to determine the impact of cellular dissociation on brain transcriptomes. We compared gene expression of microdissected samples from the dentate gyrus (DG), CA3, and CA1 subfields of the mouse hippocampus either prepared by a standard tissue homogenization protocol or subjected to enzymatic digestion used to dissociate cells within tissues. We report that compared to homogenization, enzymatic dissociation alters about 350 genes or 2% of the hippocampal transcriptome. While only a few genes canonically implicated in long-term potentiation and fear memory change expression levels in response to the dissociation procedure, these data indicate that sample preparation can affect gene expression profiles, which might confound interpretation of results depending on the research question. This study is important for the investigation of any complex tissues as research effort moves from subfield level analysis to single cell analysis of gene expression.
Highlights
Nervous systems are comprised of diverse cell types that express different genes to serve distinct functions
We found that 2.9% of the transcriptome is differentially expressed between CA1 and dentate gyrus (DG), with a roughly symmetric distribution of differential gene expression
Our analysis demonstrates that the expression of only a few cannonical long-term potentiation (LTP)-related genes is affected by the tissue prepraration method
Summary
Nervous systems are comprised of diverse cell types that express different genes to serve distinct functions. Recent advances in tissue harvesting and sequencing technologies have allowed detailed analyses of genome-scale gene expression profiles at the level of single-cell populations in the context of brain and behavior studies (Chalancon et al, 2012; Lacar et al, 2016; Mo et al, 2015; Moffitt et al, 2018; Nowakowski et al, 2018; Raj et al, 2018). These approaches have led to systems-level insights into the molecular substrates of neural function and to the discovery and validation of candidate pathways regulating physiology and behavior. The sequencing depth was different for each treatment group, this was accounted for by DESeq, which normalizes counts by sequencing depth to estimate differential gene expression
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