location: Middle region 5mm tissue treatment: NT3-chitosan(NT3) time (day): 20d tissue: spinal cord
Treatment protocol
At 1, 3, 10, 20, 30, 60, 90days post surgery, animals were sacrificed, and 5mm segments of spinal cord at the lesion site, as well as immediately caudal or rostral to the lesion segment were collected labeled with R for rostral, C for caudal, and M for lesion site
Extracted molecule
total RNA
Extraction protocol
Samples for microarray analysis were homogenized in 1 ml Trizol (Invitrogen, Carlsbad, CA), and RNA extracted using RNEasy miniprep columns (Qiagen, Valencia, CA) according to the manufacturer’s instructions.
Label
biotin
Label protocol
BiotinylatedcRNA were prepared according to the standard Affymetrix protocol from 1 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, tagged cRNA were hybridized for 16 hr at 45C on Affymetrix Rat Genome 230 2.0 Array containing 30,000 rat transcripts. The GeneChip arrays were washed and then stained (streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the GeneChip Scanner 3000.
Data processing
Data was extracted from scanned images using Affymetrix GCOS software. The raw data was normalized using a multi-array (RMA) method using the Expression Console software, followed by log2 transformation.
