|
| Status |
Public on Jun 22, 2012 |
| Title |
L5 DRG-inflamed-biological rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
inflamed L5 DRG_post-operative day 3
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley
age: adult
gender: male
tissue: lumbar dorsal root ganglion
|
| Treatment protocol |
DRG inflammation by injecting the immune activator zymosan (10 microliters, concentration 2 mg/ml, in Incomplete Freund's Adjuvant) beneath the intervertebral foramen, onto the L5 DRG on one side (PMID 16887276 and 22265726 for more details). Behavioral sensitivity was confirmed with von Frey testing. Sham animals received the same incision and intervertebral foramen exposure but no zymosan injection.
|
| Growth protocol |
single L5 DRG dissected from adult male rats 3 days after surgery for local inflammation of the DRG or sham control surgery
|
| Extracted molecule |
total RNA |
| Extraction protocol |
DRGs were placed onto dry ice then homogenized, after which RNA isolation was done using commercially available column based kits (Norgen Biotek Corp, Thorold, Ontario, Canada, Cat #24100, or Stratagene,La Jolla, CA, Cat. # 400800), including a DNA digestion step. Some samples were further concentrated by centrifugation under vacuum. Before being used for a microarray, a small aliquot from each sample was subjected to a quality control test using an Agilent Bioanalyzer to confirm that the 28S and 18S ribosomal RNA peaks were sharp, distinct, and in the proper ratio, and that the concentration and quantity were suitable for use in the microarray.
|
| Label |
biotin
|
| Label protocol |
Labeling and microarray analysis of the samples were conducted by the Gene Expression Microarray Core facility of Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. Labeling was done with the Ambion WT expression kit (Applied Biosystems) combined with the GeneChip® WT Terminal Labeling Kit (Affymetrix) to create biotin – labeled sense-strand cDNA targets for hybridization to the array
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| |
|
| Hybridization protocol |
The standard Probe Array Cartridge (GeneChip Rat Gene 1.0 Exon Array– Affymetrix) was used for hybridization of the cDNA. Hybridization was for 18 hours at 45 degrees C.
|
| Scan protocol |
Scanning was done with an Affymetrix GeneChip Scanner 3000 7G using Genechip Operating Software.
|
| Description |
INFLAMED3
|
| Data processing |
Gene-level expression analysis was conducted using GeneSpring (Agilent Technologies) version 11.5 for gene-level analysis of core transcripts. The standard workflow was used to determine genes whose expression was significantly different between sham animals and animals with local inflammation of the DRG. Probes were retained in which at least 1 sample had an expression level above a 20% cutoff, using a 0.05 p value cut-off value with the Benjamini-Hochberg correction for multiple testing, The summarization algorithm used was RMA16; the normalization method was quantile baseline transformation to median of all samples.
Probe Group File: RaEx-1_0-st-v1.r2.pgf
meta-probeset file: RaEx-1_0-st-v1.r2.dt1.rn4.core.mps
Normalized intensity values for each sample from GeneSpring version 11.5 gene level analysis of core transcripts using 20% expression level cutoff. The summarization algorithm used was RMA16; the normalization method was quantile baseline transformation to median of all samples.
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| |
|
| Submission date |
Jun 21, 2012 |
| Last update date |
Jun 22, 2012 |
| Contact name |
Judith Ann Strong |
| E-mail(s) |
judith.strong@uc.ede
|
| Phone |
513-558-2613
|
| Fax |
513-558-0995
|
| Organization name |
University of Cincinnati
|
| Department |
Anesthesiology
|
| Lab |
J-M Zhang
|
| Street address |
231 Albert Sabin Way
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45267-0531 |
| Country |
USA |
| |
|
| Platform ID |
GPL6543 |
| Series (1) |
| GSE38859 |
Expression data from pain model: rat L5 sensory ganglia after local inflammation |
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