|
| Status |
Public on Dec 15, 2012 |
| Title |
Caco2_TiO2_100_24hr-3 |
| Sample type |
RNA |
| |
|
| Source name |
Caco-2/HT29-MTX cells exposed to 100 ug/ml TiO2-NB for 24hr, biological rep3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Caco-2/HT29-MTX
cell type: human colorectal/colon adenocarcinoma
treatment: TiO2-NB
dose: 100 ug/ml
time: 24 hr
|
| Treatment protocol |
For NP exposures, 1.5 ml multi-walled carbon nanotubes (MWCNT) or titanium nanobelts (TiO2-NB) suspensions at 10 or 100 µg/ml media were placed in the top compartment (apical) of the 6 Transwell™ plate containing the 14 days co-cultured cells. The bottom compartment (basolateral) contained 2.5 ml of fresh medium. Control groups received only fresh medium in both compartments. The treated cells were incubated at 37°C – 5% CO2 for 3 and 24 h for proteomics analysis, and for 1 and 24 h for transcriptomics analysis, as described below.
|
| Growth protocol |
Caco-2, human colorectal adenocarcinoma cells (ATCC HTB-37, Lot 57863838) were maintained in EMEM media supplemented with 10% FBS (ATCC cat# 30-2003 & 30-2020) and incubated at 37°C – 5% CO2. HT29-MTX, human colon adenocarcinoma cells treated with methotrexate (Lesuffleur et al. 1993) were obtained from Dr. Thécla Lesuffleur (INSERM, Paris, France) and were maintained in DMEM with Glutamax and 10% Hi-FBS (Invitrogen) at 37°C - 5% CO2. Both cell lines were subcultured at 80-90% confluence. Caco-2 (passage 49 and 63) and HT29-MTX cells (passage 29 and 33) were cultured to obtain several Corning T75 flasks. At 80-90% confluence, the cells were trypsinized and flasks were pooled. Cells were counted using a hemocytometer and then mixed together at a 75:25 Caco-2:HT29-MTX ratio and placed in the HT29-MTX medium with 1% penicillin-streptomycin. Combined cells were mechanically mixed and 1 mL was added to 6-transwell plates to obtain 0.4 x 106 cells per well (8.9 x 104 cells/cm2) (Hilgendorf et al. 2000). Prior to NP exposure, cells were incubated at 37°C - 5% CO2 for 14 days, replacing the medium every other day, to achieve post-confluent differentiation, polarization, and tight-junction formation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was collected using the RNeasy Kit (Qiagen, Valencia, CA).
|
| Label |
biotin
|
| Label protocol |
Complementary DNA was synthesized from 3 µg of total RNA in the presence of an oligo-dT primer containing a T7 RNA polymerase promoter, and an in vitro transcription reaction was performed in the presence of a mixture of biotin-labeled ribonucleotides to produce biotinylated cRNA from the cDNA template, according to manufacturer’s protocols (Affymetrix One-Cycle Target Labeling Kit).
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| |
|
| Hybridization protocol |
Biotin-labeled cRNA (15 µg) was fragmented to a size range between 50-200 bases for array hybridization. After hybridization, the arrays were washed and stained with streptavidin-phycoerythrin.
|
| Scan protocol |
The arrays were scanned at a resolution of 2.5 microns using an Affymetrix GeneChip Scanner 3000.
|
| Description |
Gene expression data from Caco-2/HT29-MTX cells exposed to 100 ug/ml TiO2-NB for 24hr
|
| Data processing |
Raw intensity data were quantile normalized by Robust Multi-Array Analysis (RMA) summarization and probes were subject to quality control to measure the efficiency of transcription, integrity of hybridization, and consistency of qualitative calls.
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| |
|
| Submission date |
Nov 06, 2012 |
| Last update date |
Dec 15, 2012 |
| Contact name |
Susan C. Tilton |
| E-mail(s) |
Susan.Tilton@oregonstate.edu
|
| Organization name |
Oregon State University
|
| Department |
AG Envr & Molec Toxicology
|
| Street address |
1007 ALS Bldg
|
| City |
Corvallis |
| State/province |
OR |
| ZIP/Postal code |
97331 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (2) |
| GSE42066 |
Three human cell types respond to multi-walled carbon nanotubes and titanium dioxide nanobelts with cell-specific transcriptomic and proteomic expression [Caco-2/HT29-MTX cells] |
| GSE42069 |
Three human cell types respond to multi-walled carbon nanotubes and titanium dioxide nanobelts with cell-specific transcriptomic and proteomic expression |
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