Single-Cell Gene Expression Analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders

Technical and experimental advances in microaspiration techniques, RNA amplification, quantitative real-time polymerase chain reaction (qPCR), and cDNA microarray analysis have led to an increase in the number of studies of single-cell gene expression. In particular, the central nervous system (CNS) is an ideal structure to apply single-cell gene expression paradigms. Unlike an organ that is composed of one principal cell type, the brain contains a constellation of neuronal and noneuronal populations of cells. A goal is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and noneuronal cells. The unprecedented resolution afforded by single-cell RNA analysis in combination with cDNA microarrays and qPCR-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease states. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models as well as postmortem human brain tissues. This focused review illustrates the potential power of single-cell gene expression studies within the CNS in relation to neurodegenerative and neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia, respectively.     

Expression profiling using human tissues in combination with RNA amplification and microarray analysis: assessment of Langerhans cell histiocytosis

Summary. Advances in molecular genetics have led to sequencing of the human genome, and expression data is becoming available for many diverse tissues throughout the body, allowing for exciting hypothesis testing of critical concepts such as development, differentiation, homeostasis, and ultimately, disease pathogenesis. At present, an optimal methodology to assess gene expression is to evaluate single cells, either identified physiologically in living preparations, or by immunocytochemical or histochemical procedures in fixed cells in vitro or in vivo. Unfortunately, the quantity of RNA harvested from a single cell is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction (PCR) based analyses, and linear RNA amplification including amplified antisense (aRNA) RNA amplification and a newly developed terminal continuation (TC) RNA amplification methodology have been used in combination with microdissection procedures such as laser capture microdissection (LCM) to enable the use of microarray platforms within individual populations of cells obtained from a variety of human tissue sources such as biopsy-derived samples {including Langerhans cell histiocytosis (LCH)} as well as postmortem brain samples for high throughput expression profiling and related downstream genetic analyses.     

Strategies for microarray analysis of limiting amounts of RNA.

One of the critical limitations of current microarray technologies for use in expression analyses is the relatively large amount of input RNA required to generate labelled cDNA populations for array analysis. In situations where RNA is limiting, the options for expression profiling are to increase cDNA labelling and hybridisation efficiency, or to use an amplification strategy to generate enough RNA/cDNA for use with a standard labelling method. Sample amplification approaches must preserve the representation of the relative abundances of the different RNAs within the starting population and must also be highly reproducible. This review evaluates current signal and sample amplification technologies, including those that can be used to generate labelled cDNA populations for array analysis from as little as a single cell.     

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.     

RT-qPCR based quantitative analysis of gene expression in single bacterial cells

Recent evidence suggests that cell-to-cell difference at the gene expression level is an order of magnitude greater than previously thought even for isogenic bacterial populations. Such gene expression heterogeneity determines the fate of individual bacterial cells in populations and could also affect the ultimate fate of populations themselves. To quantify the heterogeneity and its biological significance, quantitative methods to measure gene expression in single bacterial cells are needed. In this work, we developed two SYBR Green-based RT-qPCR methods to determine gene expression directly in single bacterial cells. The first method involves a single-tube operation that can analyze one gene from each bacterial cell. The second method is featured by a two-stage protocol that consists of RNA isolation from a single bacterial cell and cDNA synthesis in the first stage, and qPCR in the second stage, which allows determination of expression level of multiple genes simultaneously for single bacterial cells of both gram-positive and negative. We applied the methods to stress-treated (i.e. low pH and high temperature) Escherichia coli populations. The reproducible results demonstrated that the method is sensitive enough not only for measuring cellular responses at the single-cell level, but also for revealing gene expression heterogeneity among the bacterial cells. Furthermore, our results showed that the two-stage method can reproducibly measure multiple highly expressed genes from a single E. coli cell, which exhibits important foundation for future development of a high throughput and lab-on-chips whole-genome RT-qPCR methodology for single bacterial cells.     

Localization of brain nitric oxide synthase (NOS) to human chromosome 12.

Recent research has shown that nitric oxide is a novel neuronal second messenger and transmitter that may be involved in neuronal cell death and damage in neurological illness. To map the chromosomal localization of this important brain enzyme, a rat cDNA probe was prepared by RNA PCR from rat cerebellum RNA. This rat cDNA was used to isolate a human nitric oxide synthase (NOS) cDNA from a human cerebellum cDNA library. The human cDNA clone containing 1.2 kb of brain NOS cDNA was hybridized to Southern blots containing DNAs obtained from human-rodent hybrid cell line panels using EcoRI and HindIII digestion to ascertain the location of the human NOS gene. These data showed that the human brain nitric oxide synthase mapped within 12q14-qter on human chromosome 12.     

Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance

Background  

A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20–200 micro-grams total RNA or 0.5–2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material.     

Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain

Electroporation is becoming more popular as a technique for transfecting neurons within intact tissues. One of the advantages of electroporation over other transfection techniques is the ability to precisely target an area for transfection. Here we highlight this advantage by describing methods to restrict transfection to either a single cell, clusters of cells, or to include large portions of the brain of the intact Xenopus tadpole. Electroporation is also an effective means of gene delivery in the retina. We have developed these techniques to examine the effects of regulated gene expression on various neuronal properties, including structural plasticity and synaptic transmission. Restriction of transfection to individual cells aids in imaging of neuronal morphology, while bulk cell transfection allows examination of the affects of gene expression on populations of cells by biochemical assays, imaging, and electrophysiological recording.     

Cloning of the cDNA encoding a neuronal myosin heavy chain from mammalian brain and its differential expression within the central nervous system.

The complete amino acid sequence of a neuronal myosin heavy chain (MHC) from mammalian brain (1999 amino acids, 230 kDa) has been deduced by sequencing cDNA clones isolated from a rat brain cDNA library. The library was screened using an affinity-purified polyclonal antibody that had been raised against myosin purified from a neuronally-derived cell line (Neuro-2A). Restriction digests of genomic DNA from Neuro-2A cells and rat brain are consistent with an identity of the sequenced isoform from these two sources. RNA blot analysis demonstrates this myosin to exhibit differential expression within the cerebral cortex and spinal cord. No expression was observed in liver, kidney, heart, spleen or skeletal muscle, or even within other regions of the brain. The sequence of this neuronal MHC is compared with those of other non-muscle MHCs, to which it shows an overall similarity of structure, especially with respect to conserved regions within the head (ATP binding site, actin binding site, reactive thiols) and the presence of an alpha-helical coiled-coil tail that can be arranged as 28-residue repeating units plus four skip residues. A unique non-helical tailpiece composed of 72 amino acid residues marks the C-terminus of this neuronal myosin isoform.     

Gene expression analysis of in vivo fluorescent cells

Background

The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters.

Methodology/Principal Findings

We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of ∼2∶1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR).

Conclusions/Significance

Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.