|
| Status |
Public on Dec 09, 2015 |
| Title |
CCCP Replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| 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 |
cy5
|
| Label protocol |
The RNA 6000 NanoChip kit (Agilent Technologies, number 506791511) was used with the Agilent 2100 Bioanalyzer for analysis of total RNA samples following the manual instructions. Samples with a RIN (RNA integrity number) above 7 were considered appropriate for consequent microarray testing. For Microarray analysis Agilent SurePrint G3 Human Gene Expression Microarrays (8x60K) were used. All replicates of the same treatment were labeled with the same dye (either Cy3 or Cy5). Each dye (Cy3/Cy5) had its own DMSO control.
|
| |
|
| Channel 2 |
| Source name |
C3A cells control
|
| 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 |
The RNA 6000 NanoChip kit (Agilent Technologies, number 506791511) was used with the Agilent 2100 Bioanalyzer for analysis of total RNA samples following the manual instructions. Samples with a RIN (RNA integrity number) above 7 were considered appropriate for consequent microarray testing. For Microarray analysis Agilent SurePrint G3 Human Gene Expression Microarrays (8x60K) were used. All replicates of the same treatment were labeled with the same dye (either Cy3 or Cy5). Each dye (Cy3/Cy5) had its own DMSO control.
|
| |
|
| |
| Hybridization protocol |
After hybridization, the microarrays were washed using Gene Expression Wash Buffer Kit (Agilent Technologies),
|
| Scan protocol |
Arrays were scanned using the Scan Control Software A8.5 with the feature extraction 10.X Software (Agilent technologies) on the DNA microarray scanner G2505C (Agilent Technologies).
|
| Data processing |
M values (log2FoldChange) were calculated and LOEWESS and centering normalization applied. Non uniform and control spots were removed from the data set before duplicated spots on the array were averaged (Median). Then exposure replicates were averaged using median. To identify Significantly regulated genes genes expressed |M|>2 were selected and at-test performed. Adjusted p-values were calculated using the Bonferroni-Hochberg correction and a cut-off of 0.1 applied.
|
| |
|
| 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) |
| GSE75782 |
C3A Cells: Exposure vs Control [two-channel arrays] |
| GSE75784 |
Mode of toxic action assignment of chemicals |
|
