| Autor: |
Serrano GE; Banner Sun Health Research Institute, Arizona, United States., Walker JE; Banner Sun Health Research Institute, Arizona, United States., Intorcia AJ; Banner Sun Health Research Institute, Arizona, United States., Glass MJ; Banner Sun Health Research Institute, Arizona, United States., Arce RA; Banner Sun Health Research Institute, Arizona, United States., Piras IS; Translational Genomics Research Institute, Phoenix, USA.; Institute of Arizona Alzheimer's Consortium, Phoenix, USA., Talboom JS; Translational Genomics Research Institute, Phoenix, USA.; Institute of Arizona Alzheimer's Consortium, Phoenix, USA., Nelson CM; Banner Sun Health Research Institute, Arizona, United States., Cutler BD; Banner Sun Health Research Institute, Arizona, United States., Sue LI; Banner Sun Health Research Institute, Arizona, United States., Lue LF; Banner Sun Health Research Institute, Arizona, United States., Huentelman M; Translational Genomics Research Institute, Phoenix, USA.; Institute of Arizona Alzheimer's Consortium, Phoenix, USA., Beach TG; Banner Sun Health Research Institute, Arizona, United States. |
| Abstrakt: |
Biochemical analysis of human brain tissue is typically done by homogenizing whole pieces of brain and separately characterizing the proteins, RNA, DNA, and other macromolecules within. While this has been sufficient to identify substantial changes, there is little ability to identify small changes or alterations that may occur in subsets of cells. To effectively investigate the biochemistry of disease in the brain, with its different cell types, we must first separate the cells and study them as phenotypically defined populations or even as individuals. In this project, we developed a new method for the generation of Whole Cell Dissociated Suspensions (WCDS) in fresh human brain tissue that could be shared as a resource with scientists to study single human cells or populations. Characterization of WCDS was done in paraffin-embedded sections stained with H&E, and by phenotyping with antibodies using immunohistochemistry and Fluorescence Activated Cell Sorting (FACS). Additionally, we compared extracted RNA from WCDS with RNA from adjacent intact cortical tissue, using RT-qPCR for cell-type-specific RNA for the same markers as well as whole transcriptome sequencing. More than 11,626 gene transcripts were successfully sequenced and classified using an external database either as being mainly expressed in neurons, astrocytes, microglia, oligodendrocytes, endothelial cells, or mixed (in two or more cell types). This demonstrates that we are currently capable of producing WCDS with a full representation of different brain cell types combined with RNA quality suitable for use in biochemical analysis. |
