|
| Status |
Public on Apr 03, 2013 |
| Title |
Spinal cord_lewis female_healthy control_196 |
| Sample type |
RNA |
| |
|
| Source name |
Lewis female, virgin ,healthy control
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Lewis
gender: Female
age: 16 wks
treatment: NA
days post injection: 13
tissue: spinal cord
|
| Treatment protocol |
EAE animals were treated by subcutanious injection with 50 ug of guinea-pig brain MBP (Sigma-Aldrich) in Freund's incomplete adjuvant containing 4.0 mg/ml M.Butiricium, at the base of the tail. Healthy controls were age matched untreated littermates .
|
| Growth protocol |
Rat spinal cords extracted by insuflation at the peak of disease in EAE animals and at the same time from healthy control age matched (littermates)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Spinal cords were excised from the spinal column by insufflation using a large bore blunt ended needle and 60 ml syringe. Tissue was snap frozen in liquid nitrogen prior to being stored at -80˚C. Total RNA was prepared using the Rneasy®Lipid Tissue Midi Kit (QIAGEN) and treated with DNase I to remove all traces of genomic DNA prior to storage at -80˚C . cDNA synthesis and amplification was performed using the Applause WT-Amp Plus ST kit (NuGEN).
|
| Label |
biotin
|
| Label protocol |
Samples were enzymatically fragmented and biotinylated using the Encore Biotin Module labelling kit (NuGEN)
|
| |
|
| Hybridization protocol |
5ug of fragmented biotinylated ssDNA was hybridised for 16hrs at 45oC, 60rpm on a Rat Exon 1.0 ST Array. After 16hrs GeneChips were washed on a FS_450 Fluidics Station using the washing script FS450_0001 with buffers and stains supplied with the GeneChip Hybridization, Wash, and Stain Kit from Affymetrix.
|
| Scan protocol |
Data was acquired on a 7G GeneChip Scanner 3000 and .CEL file generation performed using AGCC
|
| Description |
Total snap frozen spinal cord ,Lewis rat # 196
|
| Data processing |
Expression Console with RMA was used initially to extract probe intensity data. This data was used to access Affymetrix supplied PolyA and Eukaryotic Hybridisation controls to confirm consistent processing of the RNA samples. Subsequent analysis was performed in Partek Genomics Suite - Version 6.11.0801. The Affimetrix RaEx-1_0-st-v1.r2.dt.rn4.core.mps was used to filter probe sets. RMA background correction was applied including pre-background adjustment for CG content and quantile normalization across all chips in the experiment. Log2 transformation of probe data and mean probeset summarization was used to produce the gene-summary expression values used in the gene expression comparisons. Two way ANOVA was used to compare gene expression in the spinal cords of rats at the clinical peak of disease to age matched healthy controls.
probe group file: RaEx-1_0-st-v1.r2.pgf
meta-probeset file: RaEx-1_0-st-v1.r2.dt1.rn4.core.mps
|
| |
|
| Submission date |
Jul 05, 2012 |
| Last update date |
May 07, 2013 |
| Contact name |
Hayley Ruth Inglis |
| E-mail(s) |
h.inglis@uq.edu.au
|
| Organization name |
University of Queensland , Centre for Clinical Research
|
| Department |
Clinical Neurosciences
|
| Lab |
Neuroimmunology Group
|
| Street address |
Building 71/918, Royal Brisbane & Women’s Hospital Campus,Herston, QLD, 4029
|
| City |
Brisbane |
| State/province |
Queensland |
| ZIP/Postal code |
4029 |
| Country |
Australia |
| |
|
| Platform ID |
GPL6543 |
| Series (1) |
| GSE39119 |
Gene Expression Changes in the Spinal Cords of Lewis Rats with Myelin Basic Protein-Induced Experimental Autoimmune Encephalomyelitis (EAE) |
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