|
| Status |
Public on Oct 12, 2010 |
| Title |
rat femur on day 5 post-ablation G4.5D.16-260 |
| Sample type |
RNA |
| |
|
| Source name |
Total RNA extracted from marrow rat femur on Day 5 post-ablation
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: femoral marrow (intramedullary) tissue
strain: Sprague Dawley
age: adult
gender: male
time: day 5 post-ablation
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For three samples per time point, both the distal and proximal ends of the femurs were cut off in order to exclude the epiphyses and growth plate regions. Diaphyseal marrow tissue and cells from the mid-shaft were flushed out using Trizol®. Following homogenization, 1 ml of solution was transferred to a 1.5 ml Eppendorf tube and centrifuged at 12,000g for 10 minutes at 4°C to remove insoluble material. The supernatant containing RNA was collected, mixed with 0.2 ml of chloroform, and centrifuged at 12,000g for 15 minutes at 4°C. After RNA in the aqueous phase was transferred into a new tube, the RNA was precipitated by mixing 0.5 ml of isopropyl alcohol and recovered by centrifuging the tube at 12,000g for 10 minutes at 4°C. The RNA pellet was washed briefly in 1 ml of 75% ethanol and centrifuged at 7,500g for 5 minutes at 4°C. Finally, the total RNA pellet was dissolved in diethylpyrocarbonate (DEPC) water, and its quantity was assessed by spectrophotometric analysis.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from total RNA (GeneChip® Expression Analysis Technical Manual, 2005, Affymetrix).
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| |
|
| Hybridization protocol |
Total RNA from the samples was hybridized according to the standard Affymetrix protocol (GeneChip® Expression Analysis Technical Manual, 2005, Affymetrix), using a GeneChip® Hybridization Oven and a GeneChip® Fluidics Station.
|
| Scan protocol |
GeneChips were scanned using the GeneChip® Scanner 3000 enabled for High-Resolution Scanning. Data acquisition from the microarrays also required the use of Affymetrix GeneChip® Command Console® Software (AGCC).
|
| Description |
Gene expression data from total RNA extracted from rat femur marrow tissue on Day 5 post-ablation
|
| Data processing |
The raw gene expression data from Affymetrix were available in the form of binary CEL (Affymetrix cell intensity) files. The open-source integrated software system BRB-ArrayTools (Dr. Richard Simon and BRB-ArrayTools Development Team) was used and the CEL files were collated with RMA (robust multi-array average) method and “affy” R/Bioconductor package to compute probeset summaries. This utilized a three-step approach of background correction on PM (Perfect Match) data, quantile normalization, and Tukey’s median polish algorithm for summarization of probe level data. A univariate F-test (with random variance model) with a significance threshold of p<1×10-3 (an appropriately stringent significant level to reduce the chance of false positives) was performed with BRB-ArrayTools and used to determine differentially expressed gene probe sets over all time points. A univariate two-sample T-test with significance threshold of p<1×10-3 BRB-ArrayTools was further used to determine significant gene lists of gene probe sets differentially expression on each time point (day 1, 3, 7, 10, 14, 28, and 56 vs. day 0 intact control) post-ablation.
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| |
|
| Submission date |
Jun 14, 2010 |
| Last update date |
Oct 12, 2010 |
| Contact name |
Amarjit S. Virdi |
| E-mail(s) |
[email protected]
|
| Phone |
3129425143
|
| Fax |
3129425744
|
| URL |
http://www.rushu.rush.edu/servlet/Satellite?ProfileType=Detail&c=RushUnivFaculty&cid=1220880353240&pagename=Rush%2FRushUnivFaculty%2FFaculty_Staff_Profile_Detail_Page
|
| Organization name |
Rush University Medical Center
|
| Department |
Anatomy and Cell Biology
|
| Street address |
600 South Paulina, Armour Academic Facility, Suite 507
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60612 |
| Country |
USA |
| |
|
| Platform ID |
GPL1355 |
| Series (1) |
| GSE22321 |
Temporal Gene Expression Profiling during Rat Femoral Marrow Ablation-Induced Intramembranous Bone Regeneration |
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