Statistical Analysis of Multiplex Brain Gene Expression Images

Analysis of variance (ANOVA) was employed to investigate 9,000 gene expression patterns from brains of both normal mice and mice with a pharmacological model of Parkinson's disease (PD). The data set was obtained using voxelation, a method that allows high-throughput acquisition of 3D gene expression patterns through analysis of spatially registered voxels (cubes). This method produces multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging systems. The ANOVA model was compared to the results from singular value decomposition (SVD) by using the first 42 singular vectors of the data matrix, a number equal to the rank of the ANOVA model. The ANOVA was also compared to the results from non-parametric statistics. Lastly, images were obtained for a subset of genes that emerged from the ANOVA as significant. The results suggest that ANOVA will be a valuable framework for insights into the large number of gene expression patterns obtained from voxelation.     

Voxelation and gene expression tomography for the acquisition of 3-D gene expression maps in the brain

Voxelation and gene expression tomography or GET are novel methods for the high-throughput acquisition of gene expression patterns in the mammalian brain. Voxelation employs analysis of spatially registered voxels (cubes), while GET employs analysis of sets of parallel slices rotated about multiple independent axes of rotation. Both methods employ reconstruction of the data to result in multiple volumetric maps of gene expression analogous to those obtained from biomedical imaging techniques. Here, we describe the methodologies underlying voxelation and GET and briefly outline the insights that can be obtained from these approaches.     

Isolation of full-size mRNA from cells sorted by flow cytometry

Gene expression is one key mechanism to regulate cell growth and differentiation. It is usually determined by Northern blotting or RT-PCR. However, studies with primary cell cultures are frequently hampered due to contaminating cells such as fibroblasts. We have developed a method to isolate intact full-size mRNA from sorted cells. In many cell types, e.g. cardiac myocytes, cell sorting without prior fixation revealed complete RNA breakdown. Based on a murine fibroblast cell line (AKR-2B), ethanol and formaldehyde at various concentrations and pre-treatment with ribonuclease inactivating DEPC were compared with each other. Fixation with 75% ice-cold DEPC–pre-treated ethanol for 5 min yielded mostly intact RNA. In contrast, antibody staining prior to sorting required 15 min fixation. Addition of RNAse-free BSA (0.5%) and 2 mM CaCl₂ optimised the cell recovery ratio and thus a better RNA yield (60% compared to control) after sorting than former studies. Northern blotting and RT-PCR show the intact mRNA species β-actin. Furthermore, dependent on the cellular PCNA content, we have demonstrated the cell cycle dependent cdk2 and cyclin A expression. This fast and reliable method allows to isolate intact full-size mRNA species appropriate for Northern blotting and RT-PCR to monitor gene expression.     

High efficiency gene transfer into murine T cell clones using a retroviral vector

To establish a gene transfer and expression system for murine T cell clones, we have introduced the neomycin phosphotransferase gene encoding resistance to the neomycin analogue, G418, into non-neoplastic inducer T cell clones by using a replication-defective retroviral vector. This method allowed highly efficient gene transfer (20 to 40%) into two inducer T cell clones. The level of viral RNA expression in G418r T cells was 0.1% of poly(A)+ RNA. The infected G418r cells retained physiologic responsiveness to specific antigen as judged by antigen-specific proliferation and production of IL 3.     

Proliferation of peripheral blood mononuclear cells causes increased expression of the sodium-dependent multivitamin transporter gene and increased uptake of pantothenic acidopen star

Antigenic or mitogenic stimulation of peripheral blood mononuclear cells (PBMC) causes rapid cell proliferation. PBMC proliferation is associated with increased activities of pantothenic acid-dependent metabolic pathways, suggesting increased demand for pantothenic acid. We sought to determine whether PBMC respond to proliferation by increased cellular uptake of pantothenic acid and, if so, by what mechanism(s) the increased uptake is mediated. Uptake of pantothenic acid into PBMC was mediated by the sodium-dependent multivitamin transporter, SMVT, as judged by sodium dependency of uptake, substrate affinity and specificity, and RT-PCR of PBMC RNA. Proliferating PBMC accumulated two times more [3H]pantothenic acid than quiescent PBMC. Rates of [3H]pantothenic acid uptake paralleled rates of PBMC proliferation, as judged by uptake of [3H]thymidine. The increased uptake of [3H]pantothenic acid into proliferating PBMC was mediated by increased expression of SMVT (as judged by RT-PCR using total RNA from PBMC), leading to an increased number of transporters on the cell surface (as judged by maximal transport rates for pantothenic acid). We conclude that proliferating PBMC increase expression of the gene encoding SMVT to increase uptake of pantothenic acid.     

RNA Amplification in Brain Tissues

Recent developments in gene array technologies, specifically cDNA microarray platforms, have made it easier to try to understand the constellation of gene alterations that occur within the CNS. Unlike an organ that is comprised of one principal cell type, the brain contains a multiplicity of both neuronal (e.g., pyramidal neurons, interneurons, and others) and noneuronal (e.g., astrocytes, microglia, oligodendrocytes, and others) populations of cells. An emerging goal of modern molecular neuroscience is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subtypes and noneuronal cells. 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 amplifications, including a newly developed terminal continuation (TC) RNA amplification methodology, have been used in combination with single cell microdissection procedures to enable the use of cDNA microarray analysis within individual populations of cells obtained from postmortem brain samples as well as the brains of animal models of neurodegeneration.     

Integration of clinical and gene expression endpoints to explore furan-mediated hepatotoxicity

Molecular techniques, such as cDNA microarrays, are being used to aid in the elucidation of the mechanisms of toxicity of a variety of compounds. In this study, we evaluate the molecular effects of furan in the rat liver. Sprague-Dawley rats were exposed to 4 or 40 mg/kg furan for up to 14 days. Furan induced an initial degenerative and necrotic phenotype that was followed by inflammation and fibrosis, consistent with previous observations for this compound. RNA was harvested from each lobe of the liver at several time points to observe whether lobe-specific gene expression effects occurred. Similar gene expression changes were observed in all lobes, however the magnitude of gene expression change was more pronounced in the right lobe. Finally, to help determine the correlation between gene expression changes and liver pathology, we applied traditional microarray visualization tools to the assessment of clinical chemistry and pathology parameters.     

Laser capture microdissection of cells from plant tissues

Laser capture microdissection (LCM) is a technique by which individual cells can be harvested from tissue sections while they are viewed under the microscope, by tacking selected cells to an adhesive film with a laser beam. Harvested cells can provide DNA, RNA, and protein for the profiling of genomic characteristics, gene expression, and protein spectra from individual cell types. We have optimized LCM for a variety of plant tissues and species, permitting the harvesting of cells from paraffin sections that maintain histological detail. We show that RNA can be extracted from LCM-harvested plant cells in amount and quality that are sufficient for the comparison of RNAs among individual cell types. The linear amplification of LCM-captured RNA should permit the expression profiling of plant cell types.