|
| Status |
Public on Sep 19, 2011 |
| Title |
LPCO8A |
| Sample type |
RNA |
| |
|
| Source name |
Human mesothelial (LP9/TERT-1), 8h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LP9/TERT-1
cell type: mesothelial
treatment time: 8h
|
| Biomaterial provider |
Human mesothelial LP9/TERT-1 (LP9) cells, an hTERT-immortalized cell line phenotypically and functionally resembling normal human mesothelial cells were obtained from Dr. James Rheinwald (Dana Farber Cancer Research Institute, Boston MA).
|
| Treatment protocol |
The surface area of asbestos fibers and particles was measured using nitrogen gas sorption analysis. Fiber and particle size dimensions were determined by scanning electron microscopy. Following sterilization under ultraviolet light overnight to avoid endotoxin and microbial contamination, particulates were suspended in HBSS at 1 mg/ml, sonicated for 15 min in a water bath sonicator, and triturated 5 X through a 22-gauge needle. This suspension was added to cell medium at 0 micrometer^2 / centimeter^2 for 8h.
|
| Growth protocol |
LP9/TERT-1 cells were maintained in 50:50 DMEM/F-12 medium containing 10% fetal bovine serum (FBS), and supplemented with penicillin (50 units/ml) streptomycin (100 μg/ml), hydrocortisone (100 μg/ml), insulin (2.5 μg/ml), transferrin (2.5 μg/ml) and selenium (2.5 μg/ml).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from treated and control cells using an RNeasy Plus Mini kit according to the manufacturers protocol (Qiagen, Valencia, CA).
|
| Label |
Biotin
|
| Label protocol |
RNA samples were converted to double stranded cDNA using a standard Eberwine synthesis reaction using reverse transcriptase and T7-oligo d[T] according to the manufacturers instructions (Affymetrix, Santa Clara, CA). Following a column-based clean up of the double strand cDNA, an in-vitro transcription amplification reaction was performed to generate biotinylated-cRNA. cRNA was fragmented and prepared as an Affymetrix hybridization solution.
|
| |
|
| Hybridization protocol |
Standard Affymetrix protocols were used at 45°C, for 16h, at 60 pm
|
| Scan protocol |
Standard Affmetrix protocols were followed using GS3000.
|
| Data processing |
Robust Multi-array Average (RMA) using GeneSifter software.
|
| |
|
| Submission date |
Dec 18, 2008 |
| Last update date |
Sep 19, 2011 |
| Contact name |
Jeffrey P Bond |
| Organization name |
University of Vermont
|
| Department |
Microbiology and Molecular Genetics
|
| Street address |
95 Carrigan Dri e
|
| City |
Burlington |
| State/province |
VT |
| ZIP/Postal code |
05405 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE14034 |
Alterations in Gene Expression in Human Mesothelial Cells Correlate with Mineral Pathogenicity |
|
