|
| Status |
Public on Mar 01, 2012 |
| Title |
Rat Lung at Day 2, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Rat 8-10 wks exposed to LPS intranasally at Day 2
|
| Organism |
Rattus norvegicus |
| Characteristics |
age: 8-10 wks age
Sex: Male
strain: F344/ NCrR
genotype: wild type
|
| Treatment protocol |
For intratracheal instillations, rats were briefly anesthetized with 5% isoflurane in oxygen and instilled intranasally with 1000 μg of LPS (Pseudomonas aeruginosa serotype 10, lot 31K4122, 3,000,000 LPS units (EU)/mg, Sigma, St. Louis, MO) in 0.5 ml of 0.9% pyrogen-free saline. Control rats were not instilled.
|
| Growth protocol |
Specific pathogen-free F344/NCrR male rats of 6–8 wk of age were obtained from NCI (Frederick, MD) and were housed until 8–10 weeks of age. The rats were housed in pairs and were provided food and water ad libitum, a 12-h light/dark cycle at 22.2°C, and 30-40% humidity. Rats were weighed and randomly assigned to each experimental group.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The right lungs of F344/NCrR male rats at 0 and 2 d post LPS instillation were snap-frozen, and stored at -80 C after inflating with diluted (1:4 in PBS) Tissue-Tek O.C.T. (EMS Biosciences, Hatfeild, PA). Frozen tissue sections (8 μm thick) were fixed, dehydrated, air-dried, and epithelial cells from five large airways of each rat were captured using the laser onto Arcturus® CapSure® HS LCM Caps (Applied Biosystems, Foster City, CA). Total RNA was extracted from the cellular lysate in the cap using the PicoPure® RNA Isolation Kit (Applied Biosystems).
|
| Label |
Biotin
|
| Label protocol |
mRNA were amplified using the RiboAmp RNA amplification kit (Applied Biosystems) as per manufacturer’s instructions. Epithelia microdissected from eight tissue sections resulted in an average 1000 ng of amplified RNA. A second round of amplification was carried out and the biotinylated cRNA probes were then prepared.
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| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA (in 200 ul) were hybridized for 16 hr at 45 C on rat microarray chip RG-U34A (Affymetrix Inc., Sunnyvale, CA). Microarray Chips were washed and stained in the Affymetrix GeneChip Fluidics Station 400. Microarray Chips were washed and stained in the Affymetrix GeneChip Fluidics Station 400.
|
| Scan protocol |
Microarray Chips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
|
| Description |
Gene expression data from lung epithelial cells at Day 2 post LPS exposure
|
| Data processing |
The data were analyzed with Microarray Suite version 4.0 (MAS 4.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1500.
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| |
|
| Submission date |
Feb 29, 2012 |
| Last update date |
Mar 01, 2012 |
| Contact name |
Hitendra Chand |
| E-mail(s) |
[email protected]
|
| Phone |
505-348-8774
|
| Organization name |
Lovelace Respiraotry Research Institute
|
| Street address |
2425 Ridgecrest Dr SE
|
| City |
Albuquerque |
| State/province |
NM |
| ZIP/Postal code |
87108 |
| Country |
USA |
| |
|
| Platform ID |
GPL85 |
| Series (1) |
| GSE36174 |
LPS-induced expression in rat lung epithelial cells |
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