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.
Sforza
Daniel M
DM
Department of Molecular and Medical Pharmacology, School of Medicine, University of California, Los Angeles, California 90095-1735, USA.
Annese
Jacopo
J
Liu
Dahai
D
Levy
Shawn
S
Toga
Arthur W
AW
Smith
Desmond J
DJ
D000818:Animals; D001921:Brain / Q000033:anatomy & histology* / Q000166:cytology / Q000502:physiology; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D051379:Mice; D008810:Mice, Inbred C57BL; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification
D012313:RNA