|
| Status |
Public on Dec 31, 2016 |
| Title |
HT1080 CUR 10uM r5 |
| Sample type |
RNA |
| |
|
| Source name |
ATCC CCL121 (HT-1080 Cells)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HT-1080
cell type: Fibrosarcoma
|
| Treatment protocol |
Cells were plated in complete media containing 10% heat-inactivated FBS and allowed to attach overnight. Stock solutions of chemicals were prepared in DMSO at 1,000-fold higher concentrations than the final target doses. Stock solutions were aliquoted and stored at -20oC for up to 1 week. Dosing solutions were prepared immediately prior to treatment of the cells by diluting stock solutions 1:200 in complete media. 24 hrs after seeding the cells, the media in each well was supplemented with the dosing solutions (1:5 v/v), for a final chemical dilution of 1:1000 (0.1% vehicle). Cells were maintained in original complete media throughout the course of the experiment. Immediately following treatment, media was removed and the cells were washed with PBS and prepared for subsequent analyses
|
| Growth protocol |
HT1080 cells were grown under the conditions recommended by ATCC: Eagle’s Minimum Essential media (EMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 mg/L streptomycin and 100 U/mL of penicillin G at 37 °C in a humidified atmosphere of 95% air and 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was collected from the treated cells using the Qiaxtractor and the Qiagen VX reagent pack according to the manufacturer’s protocol (Qiagen, Valencia, CA). A DNase 1 incubation was performed on all samples to remove any possible contamination of the RNA samples with cellular DNA (Qiagen)
|
| Label |
biotin
|
| Label protocol |
RNA samples were prepared for microarray hybridization using the GeneAtlas™ 3’ IVT Express Kit according to manufacturer’s protocol (Applied Biosystems, Carlsbad, CA). Briefly, total RNA was reverse transcribed to synthesize first-strand cDNA. The cDNA was then converted into a double-stranded DNA template for transcription. Amplified cRNA (aRNA) was transcribed in vitro, with incorporation of a biotin-congugated nucleotide. The aRNA was then purified to remove unincorporated NTPs, salts, enzymes, and inorganic phosphate, and samples were fragmented to prepare the biotin-labeled aRNA samples for hybridization onto Affymetrix® GeneChip™
|
| |
|
| Hybridization protocol |
Samples were loaded onto a HT HG-U133+ PM GeneChip™ array plate and run on the GeneTitan® according to manufacturer’s protocol (Applied Biosystems, Carlsbad, CA).
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneTitan system.
|
| Description |
HT 1080 Human Fibrosarcoma Cell Line
|
| Data processing |
Analysis of gene expression data was performed using Partek software (Partek Inc., St. Louis, MO) utlising GC-RMA normalisation. A 1-way ANOVA with interactions was performed to compare gene expression between the treated and control cells. Probability values were adjusted for multiple comparisons using a false discovery rate of 5%
|
| |
|
| Submission date |
Nov 05, 2015 |
| Last update date |
Dec 31, 2016 |
| Contact name |
Rebecca A Clewell |
| Organization name |
The Hamner Institutes for Health Sciences
|
| Department |
Institute for Chemical Safety Sciences
|
| Street address |
6 Davis Drive
|
| City |
Research Triangle Park |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL13158 |
| Series (1) |
| GSE74725 |
Using transcriptomics to evaluate thresholds in genotoxicity dose-response |
|
