|
| Status |
Public on Jul 22, 2013 |
| Title |
SAEC-hTERT_saline_6month_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
SAEC-hTERT immortalized human lung small airway epithelial cell, saline only, 6 month exposed (24 passages), replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: lung
cell type: SAEC-hTERT immortalized human lung small airway epithelial cell
stress: saline
|
| Treatment protocol |
0.1 g/mL stock solutions of SWCNT, MWCNT, crocidolite asbestos and ultrafine carbon black were prepared in sterile MilliQ water containing 150 microliters/mL of the lung surfactant Survanta®. Each stock solution was sonicated for 20 seconds using a small sonication horn and then diluted in fresh culture medium and added to culture plates. Saline and dispersant only containing medium served as negative passage controls. Unexposed SAEC-hTERT cells (2x105 cells/well) were seeded to 6-well plates in triplicate per treatment (n=18). After 24 hours, cell cultures were either exposed to 0.02 micrograms/sq. cm of each particle or saline or Survanta-dispersant only for the following 6 months. Cells were passage matched for a total of 24 passages. Exposed media was changed every 3 to 4 days while cells were split once every 7 days.
|
| Growth protocol |
Cells were maintained in SABM medium (Lonza) supplemented with SAGM SingleQuot (Clonetics) growth factor kit and 100 U/mL penicillin and streptomycin. Cell cultures were held in a humid, 37o C and 5% CO2 cell culture incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA samples from each replicate (total n=18) were collected with TRIzol (Invitrogen) post-exposure using manufactorer's protocol. RNA quality was assessed in house using gel electrophoresis and 260/280 nm absorbance ratio. Samples stored at -80o C were shipped to ArrayStar (Rockville, MD). At ArrayStar, samples were treated with DNase digestion and analyzed for RNA quality using Nanodrop ND-100 and Bioanalyser 2100.
|
| Label |
Cy3
|
| Label protocol |
cDNA samples were amplified, Cy3-labeled with NimbleGen one-color DNA labeling kit following manufactorer's protocol. Microarray hybridization was performed at 65°C for 17 hours to NimbleGen whole human genome 12x135K microarrays in NimbleGen Hybridization System.
|
| |
|
| Hybridization protocol |
Microarray hybridization was performed at 65°C for 17 hours on NimbleGen 12x135k Whole Human Genome Oligonucleotide Microarray (HG18) in NimbleGen Hybridization System.
|
| Scan protocol |
After being washed in an ozone-free environment, image capture was performed with Axon GenePix 4000B (Molecular Devices) and uploaded into NimbleScan (v2.5), and Cy3 label intensities determined.
|
| Description |
Genome expression after 6 month saline-only in vitro exposure
Sample name: H-SAL 3
|
| Data processing |
GenePix data were uploaded into NimbleScan for Cy3 label intensity determination. Intensities were quantile normalized using Robust Multichip Average method included in NimbleScan. Only genes expressing ≥50.0 normalized intensity in all 18 samples were subjected to further analysis. Gene expression text files were imported into GeneSpring GX (ver. 10.0) for further analysis.
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| |
|
| Submission date |
Sep 27, 2012 |
| Last update date |
Jul 22, 2013 |
| Contact name |
Todd A Stueckle |
| E-mail(s) |
tstueckle@cdc.gov
|
| Phone |
304 285-6098
|
| Organization name |
National Institute for Occupational Safety and Health
|
| Department |
Health Effects Laboratory Division
|
| Lab |
Pathology and Physiology Research Branch
|
| Street address |
1095 Willowdale Road
|
| City |
Morgantown |
| State/province |
WV |
| ZIP/Postal code |
26505 |
| Country |
USA |
| |
|
| Platform ID |
GPL16025 |
| Series (1) |
| GSE41178 |
Whole genome profile of SWNCT and MWCNT vs. asbestos subchronic exposures to human small airway epithelial cells |
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