|
| Status |
Public on Dec 09, 2015 |
| Title |
FCCP Replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
C3A cells exposed 2h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: C3A
|
| Treatment protocol |
C3A cells were exposed for 2 hours to substances. For microarray analysis, 6 replicate wells per concentration were pooled. DMSO (0.1%) was used as solvent control.
|
| Growth protocol |
C3A cells were grown in 75 cm2 culture flasks until approx. 80% confluency. Cells were then subcultured as quoted above until the uptake of trypsinised cells in media. C3A cells were diluted to reach a final density of 250 000 cells/ml. 200 µl of cell suspension were seeded in the inner 60 wells of a black 96-well plate with clear bottom (Greiner bio-one, Product 655906, Reinach, Switzerland). The outer rows were filled with 200 µl 1x PBS (phosphate buffered saline, Sigma-Aldrich, Buchs, Switzerland). Cells were pre-incubated at 37°C for 24 h. After pre-incubation cells were dosed with the test chemicals.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy Mini Kit (QUIAGEN, Product 74104, Hombrechtikon, Switzerland) according to manufacturers’ instructions. DNA was digested using the RNeasy Mini Kit optional on-column DNase digestion. Quality of extracted RNA was checked via the Nanodrop 1000 spectraphotomoeter V3.7, as well as running the samples on a 1.5% agarose gel.
|
| Label |
cy3
|
| Label protocol |
For Microarray analysis, Agilent SurePrint G3 Human Gene Expression Microarrays (8x60K) were used in combination with a one-color based hybridization protocol (OneColor RNA Spike-In Mix, Agilent Technologies, number 51885282). All steps were carried out according to the manufacturer´s instructions.
|
| |
|
| Hybridization protocol |
After hybridization, the microarrays were washed using Gene Expression Wash Buffer Kit (Agilent Technologies),
|
| Scan protocol |
Fluorescent signal intensities were detected with Scan Control A.8.4.1 Software (Agilent Technologies) on the Agilent DNA Microarray Scanner and extracted from the images using Feature Extraction 10.7.3.1 Software (Agilent Technologies).
|
| Data processing |
The data was normalized using quantile-normalization. Control spots as well as not uniform spots were removed. The solvent control was averaged and the M value (log2FoldChange) calculated. Duplicated spots on the array were averaged before exposure replicates were averaged using the median.
|
| |
|
| Submission date |
Dec 08, 2015 |
| Last update date |
Dec 09, 2015 |
| Contact name |
Jessica Legradi |
| E-mail(s) |
jessica.legradi@vu.nl
|
| Organization name |
VU Amsterdam
|
| Department |
IVM
|
| Street address |
De Boelelaan 1087
|
| City |
Amsterdam |
| ZIP/Postal code |
1081 HV |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL13607 |
| Series (2) |
| GSE75783 |
C3A Cells: Exposure vs Control [one-channel arrays] |
| GSE75784 |
Mode of toxic action assignment of chemicals |
|
