|
| Status |
Public on Aug 01, 2011 |
| Title |
CF CFTR unexposed replicate 4 |
| Sample type |
RNA |
| |
|
| Source name |
CF CFTR unexposed
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: CFBE41o- ∆F508-CFTR
|
| Treatment protocol |
PAO1 was added to the apical side of monolayers at a multiplicity of infection (MOI) of 30:1 for 1 hour in the absence of antibiotics, and then planktonic PAO1 was removed by replacing the apical medium with MEM supplemented with 0.4% arginine. Control monolayers were treated identically except that vehicle only (medium used to grow PAO1, MEM supplemented with 0.4% arginine) was added to the apical side of cells.
|
| Growth protocol |
CFBE41o- cells, a human bronchial epithelial cell line homozygous for the ∆F508-CFTR mutation, and CFBE41o- cells complemented with either ∆F508-CFTR (CFBE-∆F508-CFTR) or wt-CFTR (CFBE-wt-CFTR) were generously provided by Dr. J.P. Clancy (University of Alabama). In all studies, we examined the effect of P. aeruginosa strain PAO1 on CFBE-∆F508-CFTR cells compared to CFBE-wt-CFTR cells, since both lines contain three copies of CFTR (either ∆F508/∆F508/∆F508 or ∆F508/∆F508/wt-CFTR). Cells were grown as polarized monolayers in air-liquid interface culture on Transwell filters as described previously. Briefly, cells were seeded at 1 x 106 on 24 mm Transwell filters and grown for 8 days (to develop polarized monolayers) in minimal essential medium (MEM) (Mediatech, Herndon, VA) with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 g/ml streptomycin at 37°C and 5% CO2, balance air.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were then homogenized with 600 μl RLT lysis buffer from the Qiagen RNeasy kit, and the homogenate was vortexed for several seconds and drawn through a 20-gauge needle 10 times. We then added 600 μl of 100% ethanol, and applied samples to an RNeasy column for separation following manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
First-strand cDNA synthesis was conducted by priming the mRNA with a T7-(dT24) primer. Double-stranded cDNA was prepared using Life Technology Superscript cDNA Synthesis System (Invitrogen, Carlsbad, CA). From cDNA, cRNA was synthesized using the T7 MegaScript In Vitro Transcription kit (Ambion, Austin, TX). Synthesized cRNA was labeled during transcription (Megascript system; Ambion) with biotin-11–cytidine triphosphate and biotin-16–uridine triphosphate (Enzo Diagnostics, Farmingdale, NY).
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| |
|
| Hybridization protocol |
Biotinylated cRNA products were hybridized to Affymetrix genechip HGU 133Plus2 arrays for 16 h at 40°C in a Gene Chip Hybridization oven 640 using the manufacturer's hybridization buffer (Affymetrix, Santa Clara, CA) in the Dartmouth Genomics and Microarray Laboratory (http://dms.dartmouth.edu/dgml/background/).
|
| Scan protocol |
Arrays were washed and stained using the GeneChip® Fluidics Station 450 (Affymetrix). The chips were scanned with the 7G Affymetrix Gene Scanner (Affymetrix) according to manufacturer's procedures.
|
| Description |
GT051310_Sample Delta F -4.CEL
|
| Data processing |
Raw fluorescence values were normalized and summarized raw gene array data using RMA as implemented in Bioconductor.
|
| |
|
| Submission date |
Jul 06, 2011 |
| Last update date |
Aug 01, 2011 |
| Contact name |
Thomas H Hampton |
| E-mail(s) |
[email protected]
|
| Phone |
603 650-1184
|
| URL |
http://www.dartmouth.edu/~toxmetal/about/research-team/faculty/tom.html
|
| Organization name |
Dartmouth Medical School
|
| Department |
Pharm/Tox
|
| Lab |
Bruce Stanton
|
| Street address |
North College Street
|
| City |
Hanover |
| State/province |
NH |
| ZIP/Postal code |
03755 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE30439 |
Exposure of cystic fibrosis bronchial epithelial cells (CFBE 41 o-) to Pseudomonas aeruginosa (PA01) biofilms |
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