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Series GSE4037

Query DataSets for GSE4037

Status

Public on Jan 13, 2006

Title

prosc-affy-rat-185420

Organism

Rattus norvegicus

Experiment type

Expression profiling by array

Summary

The goal of the research proposed herein is to identify (i) gene products that allow identification of specific precursor populations in vivo and (ii) genetic programs that regulate the generation of lineaege-restricted precursor (LRP) cells from neuroepithelial stem cells (NSC). As these cells play a major role in development and tissue repair, this effort will enhance both basic and clinically relevant research. Note: Funding for this micro-array analysis was granted through NINDS as supplement 3R01NS042820-02S1.
Our ability to generate essentially unlimited numbers of purified rodent NSCs, thier restricted decendants leading the generation of macroglia - and also to control the generation of each of these cell types from its immediate ancestor - provides us with a unique system for studying the multi-step nature of differentiation. Using defined conditions, samples will be isolated from each type of purified cell population. Expression profiles will be compared to reveal cell type specific markers and regulatory factors.
The generation of differentiated cell types from neuroepithelial stem cells appears to require progression through a sequence of lineaege-restricted intermediate cell stages. In the case of at least one of the pathways involved in oliigodendrocyte generation, it now appears that all of the stages of this sequence have been defined and all of the intermediate cells have been isolated (as detailed in the Noble, M. et al. (2004) Dev. Biol. 265, 33-52). Studies in vitro, and consequent to cell transplantation, have enabled us to gain an increasingly detailed understanding of the differentiation potential of these cells. The microarray analysis proposed herein will provide the specific markers that will enable the LRP cells to be unambiguously identified in vivo, thereby setting the stage to develop a detailed mapping of the role of these cells during development.
RNA will be isolated from purified cell populations. All populations will initially be derived from spinal cord. Preparation of purified populations of each of these cell types has been described in multiple of our previous publications (Mayer, M. et al. (1994) Development 120, 142-153; Mayer-Pröschel, M. et al. (1997) Neuron 19, 773-785; Rao, M. and Mayer-Pröschel, M. (1997) Dev. Biology 188, 48-63). Total RNA from purified cell cultures will be extracted in a two step process. RNA will first be extracted by dounce homogenization in phenol/guanidine isothiocyonate solution (Trizol, Invitrogen, California) followed by isopropanol precipitation. Contaminating traces of DNA and protein will then be removed by silica-based affinity chromatography and Dnase treatment (NucleoSpin RNA Purification system, Clontech, California). Depending on the cell type, 5x105 cells will typically yield 5-15µg of high quality RNA (OD260/280=2.1 at pH7.5). In order to determine purity and molecular weight distribution, samples will be routinely tested on a Agilent Bioanalyzer. cDNA will be generated from total RNA and then be submitted to one round of amplification and labeling using the messageAmp system (Ambion). Probes are then hybridized to the Affymerix Rat 230 (A/B or 2.0) GeneChip expression array. Array data will be processed using the Micro Array Suite 5.0 (MAS, Affymetrix). Cel files containing normalized, relative expression data for all genes tested will be used for downstream statistical analysis.
Keywords: other

Contributor(s)

Proschel C

Citation missing

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Submission date

Jan 13, 2006

Last update date

Jul 31, 2017

Contact name

Winnie Liang

E-mail(s)

[email protected]