High throughput analysis of the human and mouse spinal cord neural stem cell niche

Anamniotes, rodents and man maintain a pool of adult neural stem cells around the central canal in the spinal cord representing an attractive cellular source for endogenous repair. Cell diversity and genes specific for this niche are still ill-defined in mammals. To identify genes specifically expressed in the niche, we microdissected (with laser) the central canal region and the adjacent tissue in human and mice adult tissues. Total RNA was isolated and used to probe affymetrix microarrays We microdissected 2 human (aged 17 and 46) and 4 mouse (3 months old) samples. Central canal and adjacent tissues were microdissected with PALM micro beam equipment. RNA extraction was performed using the ReliaPrep RNA cell Miniprep System (Promega, Madison, USA) . RNA profiling was performed using affymetrix microarray technology.Hybridization targets were obtained following a double amplification procedure according to the protocol developed by Affymetrix (GeneChipTwo-Cycle Eukaryotic Target Labeling Assay; Affymetrix, USA). A hybridization mixture containing 10 µg of biotinylated cRNA was generated. The biotinylated cRNA was hybridized to HT_HG-U133_Plus_PM (human) and HT_MG-430_PM (mouse) Affymetrix microarrays. Four mouse and two human samples (EZ and periEZ regions each) were analyzed in total. The microarrays were scanned using the Affymetrix Gene Atlas scanner.The data files were generated with Affymetrix Expression Console v1.2.1 and gene expression data were normalized with GC-RMA algorithm.