|
| Status |
Public on Mar 01, 2014 |
| Title |
Daphnia_copper_96h_CuO3_REF |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
CuO exposure sample; whole Daphnia
|
| Organism |
Daphnia magna |
| Characteristics |
age when sampled: 96 hours
tissue: whole body extracts
|
| Treatment protocol |
96 hours of exposure to EC10 (immobilization) concentrations of nanoparticles, salts or not exposed (blank); medium refreshment and exposure to fresh spike of nanoparticles/salt after 48 hours
|
| Growth protocol |
Exposed in ISO test medium (CaCl2.2H2O: 0.294 g/l, MgSO4.7H2O: 0.123 g/l, NaHCO3: 0.065 g/l KCl: 0.006 g/l) for 96 hours, fed on algae species Pseudokirchneriella subcapitata and Chlamydomonas reinhardtti (in a 3:1 ratio)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each replicate, the RNA of pooled samples (40 daphnids) was extracted using the Trizol RNA extraction method (Invitrogen), followed by a DNAse treatment (Fermentas) and phenol/chloroform extractions.
|
| Label |
cy 5
|
| Label protocol |
The Agilent Low Input Quick Amp Labeling Kit was used to convert the total RNA (100 ng) to amplified labeled cRNA according to the two-color microarray-based gene expression analysis protocol (version 6.5, Agilent Technologies). For each exposure experiment (ZnO, ZnCl2, Blank on the one hand and CuO, CuCl2.2H2O, Blank on the other hand) a reference design with swapped dyes (to avoid dye bias) was used. Two replicates of each exposure condition (metal oxide, salt, blank) were labeled with the fluorescent dye cyanine 3 (cy3) and two other replicates were labeled with the fluorescent dye cyanine 5 (cy5), while the reference sample was labeled with both cy3 and cy5. The labeled cRNA was purified using RNeasy mini spin columns (Qiagen). The quantity (yield) and labeling efficiency (specific activity) of these cRNA samples was checked with Nanodrop, by measuring the RNA concentration (absorbance at 260 nm) and the cy3 (absorbance at 550 nm) and cy5 (absorbance at 650 nm) incorporation.
|
| |
|
| Channel 2 |
| Source name |
Copper reference sample; whole Daphnia
|
| Organism |
Daphnia magna |
| Characteristics |
age when sampled: 96 hours
tissue: whole body extracts
|
| Treatment protocol |
96 hours of exposure to EC10 (immobilization) concentrations of nanoparticles, salts or not exposed (blank); medium refreshment and exposure to fresh spike of nanoparticles/salt after 48 hours
|
| Growth protocol |
Exposed in ISO test medium (CaCl2.2H2O: 0.294 g/l, MgSO4.7H2O: 0.123 g/l, NaHCO3: 0.065 g/l KCl: 0.006 g/l) for 96 hours, fed on algae species Pseudokirchneriella subcapitata and Chlamydomonas reinhardtti (in a 3:1 ratio)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each replicate, the RNA of pooled samples (40 daphnids) was extracted using the Trizol RNA extraction method (Invitrogen), followed by a DNAse treatment (Fermentas) and phenol/chloroform extractions.
|
| Label |
cy 3
|
| Label protocol |
The Agilent Low Input Quick Amp Labeling Kit was used to convert the total RNA (100 ng) to amplified labeled cRNA according to the two-color microarray-based gene expression analysis protocol (version 6.5, Agilent Technologies). For each exposure experiment (ZnO, ZnCl2, Blank on the one hand and CuO, CuCl2.2H2O, Blank on the other hand) a reference design with swapped dyes (to avoid dye bias) was used. Two replicates of each exposure condition (metal oxide, salt, blank) were labeled with the fluorescent dye cyanine 3 (cy3) and two other replicates were labeled with the fluorescent dye cyanine 5 (cy5), while the reference sample was labeled with both cy3 and cy5. The labeled cRNA was purified using RNeasy mini spin columns (Qiagen). The quantity (yield) and labeling efficiency (specific activity) of these cRNA samples was checked with Nanodrop, by measuring the RNA concentration (absorbance at 260 nm) and the cy3 (absorbance at 550 nm) and cy5 (absorbance at 650 nm) incorporation.
|
| |
|
| |
| Hybridization protocol |
300 ng of a labeled cy3 or cy5 cRNA sample from the exposure was co-hybridized with a cy5 or cy3 cRNA reference sample on a 15 K Daphnia magna microarray (Agilent-023710, Platform GPL16579), for the different replicates, at 60°C during 17 h in a rotating hybridization oven (Agilent G2545A). The arrays were washed according to the two-color microarray-based gene expression analysis protocol (version 6.5, Agilent Technologies).
|
| Scan protocol |
The arrays were scanned with Genepix personal 4100a scanner (Axon Instruments) at 532 (cy3) and 635 (cy5) nm with a resolution of 5 µm and adjusted photomultiplier tube (PMT) values to obtain a ratio cy5/cy3 of around 1 (0.98 < ratio < 1.02).
|
| Data processing |
The statistical analysis of the microarray data was performed with the R package limma. Spots for which red or green FG < BG + 2SD on all arrays, were deleted (FG: median foreground intensity, BG: average local background intensity calculated over the full microarray, SD: standard deviation of local background intensities). Subsequently, the remaining data were vsn normalized and background corrected (by subtracting the local background from the foreground for each spot). Linear models were fitted to intensity ratios, after which probes were ranked in order of evidence of differential expression using an empirical Bayes method. The Zn exposures (ZnO nanoparticles and ZnCl2) were contrasted against their controls and against each other. These contrasts were fitted to the linear model and considered significantly differentially expressed genes if false discovery rate (FDR) < 0.1 and |log2FC| > 0.585 (log2 fold change, corresponds to 1.5 fold change). The same analysis was done for the Cu exposures.
|
| |
|
| Submission date |
Feb 06, 2014 |
| Last update date |
Mar 01, 2014 |
| Contact name |
Nathalie Adam |
| E-mail(s) |
Nathalie.adam@uantwerpen.be
|
| Organization name |
University of Antwerp
|
| Department |
Biology
|
| Lab |
Systemic Physiological and Ecotoxicological Research
|
| Street address |
Groenenborgerlaan 171
|
| City |
Antwerp |
| ZIP/Postal code |
2020 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL16579 |
| Series (2) |
| GSE54738 |
The effect of CuO nanoparticles on gene expression patterns in Daphnia magna |
| GSE54739 |
The effect of ZnO and CuO nanoparticles on gene expression patterns in Daphnia magna |
|
