Single-Cell Microarray Analysis in Hippocampus CA1: Demonstration and Validation of Cellular Heterogeneity

Abstract

Laser capture microdissection in combination with microarrays allows for the expression analysis of thousands of genes in selected cells. Here we describe single-cell gene expression profiling of CA1 neurons in the rat hippocampus using a combination of laser capture, T7 RNA amplification, and cDNA microarray analysis. Subsequent cluster analysis of the microarray data identified two different cell types: pyramidal neurons and an interneuron. Cluster analysis also revealed differences among the pyramidal neurons, indicating that even a single cell type in vivo is not a homogeneous population of cells at the gene expression level. Microarray data were confirmed by quantitative RT-PCR and in situ hybridization. We also report on the reproducibility and sensitivity of this combination of methods. Single-cell gene expression profiling offers a powerful tool to tackle the complexity of the mammalian brain.

Methodological Considerations Regarding Single-Cell Gene Expression Profiling for Brain Injury

“…21,[34][35][36] Instead, it combines all of the different cell types present in a sample tissue (neurons, glia, blood cells and vessels, and so forth) at potentially disparate stages of injury and disease processes. [25][26][27][28][29][30][31][32][33] As a result, the gene expression profiles of selectively vulnerable subpopulations of cells may be largely obscured, and the detection of potentially important, less abundantly expressed genes (such as transcription factors) may be overlooked.…”

“…Several studies have exploited this capacity to characterize changes in gene expression among specific subpopulations of cells and cell types under both normal and pathological conditions (Table II). 2,[21][22][39][40][41][42][43] An investigator may also choose to pool multiple cells of a common phenotype or those that meet specific criteria in order to account for normal biological variability between individual cells and/or animals. 19,[41][42] In order to ensure that enough genetic material is available for subsequent gene expression analysis, amplification of the RNA obtained from a single cell is required.…”

“…This linearity can be attributed to the highly efficient T7 polymerase used for amplification and has been examined by several groups using microarrays, Northern blotting, and RT-PCR. 21,36,45 The basic aRNA amplification protocol involves first incorporating a specialized oligo-dT primer that contains the sequence of a T7 RNA polymerase promoter into the complementary DNA of the sample mRNAs. Many copies are then generated from each original cDNA in a subsequent RNA polymerase reaction (Fig.…”

“…48 A shortcoming to the latter LCM method is that it requires the use of unfixed tissue, introducing a greater potential for contamination by active endogenous RNases. Kamme et al 21 recently demonstrated LCM along with aRNA amplification and cDNA microarray analysis in a study designed to evaluate cellular heterogeneity in the CA1 region of the rat hippocampus. Both microdissection methods are effective and share similar stipulations regarding the contamination of a sample from neighboring cells and tissue.…”

“…20 In situ hybridization also offers the added benefit of providing information about the localization of individual mRNAs. 21,22 Together, the combination of genomic microarrays and traditional molecular biology techniques provides a reliable and powerful set of tools to detect patterns of differential gene expression in the brain and other tissues. Techniques such as immunohistochemistry, Western blotting, and EnzymeLinked Immunosorbent Assay (ELISA) can then be used to analyze more directly individual protein levels and localization.…”