Sabina Berl1, Khalad Karram1, Anja Scheller2, Melanie Jungblut3, Frank Kirchhoff2, Ari Waisman4. 1. Institute for Molecular Medicine, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. 2. Center for Integrative Physiology and Molecular Medicine (CIPMM), Molecular Physiology, University of Saarland, Building 48, D-66421 Homburg, Germany. 3. Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, D-51429 Bergisch-Gladbach, Germany. 4. Institute for Molecular Medicine, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. Electronic address: waisman@uni-mainz.de.
Abstract
BACKGROUND: Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases. NEW METHODS: Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads. RESULTS: Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3-5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation. COMPARISON WITH EXISTING METHODS: Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression. CONCLUSION: These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS.
BACKGROUND: Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases. NEW METHODS: Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads. RESULTS: Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3-5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation. COMPARISON WITH EXISTING METHODS: Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression. CONCLUSION: These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS.
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