|
| Status |
Public on Nov 30, 2010 |
| Title |
mouse540-lungs-C57BL/6J Treated-rep-3 |
| Sample type |
RNA |
| |
|
| Source name |
C57BL/6J Treated
|
| Organism |
Mus musculus |
| Characteristics |
gender: male
tissue: whole lung
strain: C57BL/6J
|
| Treatment protocol |
Control mice were i.p. sensitized with 10 ug Der p 1 on days 1 and 8, then challenged with PBS on day 15. Treated mice were i.p. sensitized with 10 ug Der p 1 days 1 and 8 and then challenged with 50 ug Der p 1 on day 15. All mice were harvested on day 18 (72 hours after challenge).
|
| Growth protocol |
The right lower lobe was inflated with OCT and then flash frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lung lobes were homogenized and then RNA was isolated using RNeasy Mini Kits (Qiagen), according to the manufacturer’s instructions. RNAs were DNAseI treated, then rRNAs were removed using Invitrogen RiboMinus kits.
|
| Label |
biotin
|
| Label protocol |
RNA quality and quantity was ensured using the Bioanalyzer (Agilent, Inc) and NanoDrop (Thermo Scientific, Inc) respectively. Per RNA labeling, 1 microgram of total RNA was used in conjunction with the Affymetrix recommended protocol for the GeneChip® WT Sense Target Labeling. The total RNA was first subjected to a ribosomal RNA reduction using the Invitorgen RiboMinus Transcriptome Isolation Kit (catalog# K1550-01). The remaining rRNA striped RNA is reverse transcribed in the presence of T7(N6) primers and a double stranded cDNA is generated. An In Vitro Transcription is performed and the resulting cRNA is purified. A first strand cDNA is generated form the cRNA in the presence of dUTPs. After RNA hydrolysis, the single stranded cDNA is fragmented and end labeled with Biotin.
|
| |
|
| Hybridization protocol |
The hybridization cocktail containing the fragmented and Biotin labeled cDNAs were hybridized to The Affymetrix GeneChip® Mouse Exon 1.0 ST Array. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols as described by Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Molecular Probes, Carlsbad, CA) and enhanced by using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA).
|
| Scan protocol |
An Affymetrix Gene Chip Scanner 3000 was used to scan the probe arrays. Gene expression intensities were calculated using the Affymetrix Expression Console software and the Cel files generated.
|
| Description |
RMA expression value derived from Partek Genomics Suite software; core-exon analysis
|
| Data processing |
Raw data were processed and analyzed using Partek Genomics Suite Software. Core-exon analysis was used and then gene-level estimates of expression were calculated.
|
| |
|
| Submission date |
Nov 30, 2009 |
| Last update date |
Nov 30, 2009 |
| Contact name |
Christopher Pan |
| E-mail(s) |
cpan@mail.nih.gov
|
| Organization name |
NIH
|
| Department |
NHGRI
|
| Street address |
Rm 5228, Bldg 50, South Dr.
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL6096 |
| Series (1) |
| GSE19223 |
Strain-dependent Dissociation of Airway Hyper-responsiveness from Inflammation in a House Dust Mite Model of Asthma |
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