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.     

Anatomical Methods for Voxelation of the Mammalian Brain

Voxelation allows high-throughput acquisition of three-dimensional gene expression patterns in the brain through analysis of spatially registered voxels (cubes). The method results in multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging techniques. An important issue for voxelation is the development of approaches to anchor correctly harvested voxels to the underlying anatomy. Here, we describe experiments to identify fixation and cryopreservation protocols for improved registration of harvested voxels with neuroanatomical structures. Paraformaldehyde fixation greatly reduced RNA recovery as judged by ribosomal RNA abundance. However, gene expression signals from paraformaldehyde-fixed samples were not appreciably diminished as judged by average signal-noise ratios from microarrays, highlighting the difficulties of accurate quantitation of cross-linked RNA. Additional use of cryoprotection helped to improve further RNA recovery and signal from fixed tissue. It appears that the best protocol to provide the necessary resolution of neuroanatomical information in voxelation entails a controlled dose of fixation and thorough cryoprotection, complemented by histological staining.