|
| Status |
Public on Sep 01, 2013 |
| Title |
ref_par_48_2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
reference sample
|
| Organism |
Daphnia magna |
| Characteristics |
clone: P 32,85
gender: female
treatment: reference
|
| Treatment protocol |
Eighty juveniles per treatment were kept together in 500ml jars at a density of 1 animal/2.5ml medium and exposed to four conditions (two biological replicates per treatment): a control treatment without stress, fish kairomones (Leuciscus idus), carbaryl (5.6µg/l; measured concentration 5.74µg/l) or spores of the parasite Pasteuria ramosa (5x10^6 spores/ml).
Exposures were done every second day starting at day 0. Medium was completely renewed at the moment of a new stress pulse. The two biological samples were collected after 48h, 96h and 144h of exposure. The last pulse was given three hours before freezing the animals for gene expression analysis, respectively, on 45h-, 93h- and 141h-old Daphnia.
|
| Growth protocol |
P 32,85 clone cultured in standard conditions to avoid maternal effects (see Jansen et al.).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following the manufacturer's protocol.
|
| Label |
Cy5
|
| Label protocol |
cDNA collection and sample labelling were done according to Soetaert et al. (2007) and Vandenbrouck et al. (2009). Briefly, total RNA (5µg) together with 2µl mRNA spike from the Lucidea Universal Scorecard (Amersham Biosciences) or from the Spotreport Alien cDNA Array Validation System (Agilent Technologies' Stratagene products) was converted into cDNA using random primers (Invitrogen), a dNTP labelling mix containing aminoallyl-dUTPs and 400U Superscript II reverse transcriptase (Invitrogen). Cy5 and Cy3 (Amersham Biosciences) were covalently coupled to the aminoallyl cDNA samples. After a cleanup reaction (QIAquick PCR purification kit, Qiagen, USA) removing all non-incorporated dyes, the quality of the labelled cDNA was checked spectrophotometrically using the Nanodrop. A total amount of 50 pmol labelled cDNA with a frequency of incorporation between 20 and 50 was vacuum dried to use as probe.
|
| |
|
| Channel 2 |
| Source name |
parasite exposed, 48h, r2
|
| Organism |
Daphnia magna |
| Characteristics |
clone: P 32,85
gender: female
treatment: parasite exposure
time point: 48h
replicate: 2
|
| Treatment protocol |
Eighty juveniles per treatment were kept together in 500ml jars at a density of 1 animal/2.5ml medium and exposed to four conditions (two biological replicates per treatment): a control treatment without stress, fish kairomones (Leuciscus idus), carbaryl (5.6µg/l; measured concentration 5.74µg/l) or spores of the parasite Pasteuria ramosa (5x10^6 spores/ml).
Exposures were done every second day starting at day 0. Medium was completely renewed at the moment of a new stress pulse. The two biological samples were collected after 48h, 96h and 144h of exposure. The last pulse was given three hours before freezing the animals for gene expression analysis, respectively, on 45h-, 93h- and 141h-old Daphnia.
|
| Growth protocol |
P 32,85 clone cultured in standard conditions to avoid maternal effects (see Jansen et al.).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol following the manufacturer's protocol.
|
| Label |
Cy3
|
| Label protocol |
cDNA collection and sample labelling were done according to Soetaert et al. (2007) and Vandenbrouck et al. (2009). Briefly, total RNA (5µg) together with 2µl mRNA spike from the Lucidea Universal Scorecard (Amersham Biosciences) or from the Spotreport Alien cDNA Array Validation System (Agilent Technologies' Stratagene products) was converted into cDNA using random primers (Invitrogen), a dNTP labelling mix containing aminoallyl-dUTPs and 400U Superscript II reverse transcriptase (Invitrogen). Cy5 and Cy3 (Amersham Biosciences) were covalently coupled to the aminoallyl cDNA samples. After a cleanup reaction (QIAquick PCR purification kit, Qiagen, USA) removing all non-incorporated dyes, the quality of the labelled cDNA was checked spectrophotometrically using the Nanodrop. A total amount of 50 pmol labelled cDNA with a frequency of incorporation between 20 and 50 was vacuum dried to use as probe.
|
| |
|
| |
| Hybridization protocol |
Arrays were hybridized overnight at 42°C. See Vandenbrouck et al. 2009 (PMID 19187980) for more details.
|
| Scan protocol |
Genepix personal 4100 Scanner and Genepix pro 4.1 software (Axon Instruments).
|
| Description |
14_010409
This Sample uses the array with Lucidea controls.
|
| Data processing |
Statistical analysis was performed using the R package limma (Smyth, 2004). Spots for which red or green FG < BG + 2SD (Sclep et al., 2007) on all arrays were deleted before analysis (FG: median foreground intensity, BG: average local background intensity calculated over the full microarray, SD: standard deviation of local background intensities). Median intensity data were background corrected using a normal-exponential convolution model using the function backgroundCorrect with method 'normexp', offset = 50 (Ritchie et al., 2007). Data were loess-normalized using the function normalizeWithinArrays, method = 'loess' (Smyth & Speed, 2003). Linear models were fitted to intensity ratios, after which probes were ranked in order of evidence of differential expression using an empirical Bayes method (Smyth, 2004). Each stressor was contrasted against the controls. These contrasts were fitted to the linear models and cut off at p < 0.05 and log2FC > 0.75 or log2FC < -0.75 (log2 fold change).
|
| |
|
| Submission date |
Sep 23, 2010 |
| Last update date |
Sep 01, 2013 |
| Contact name |
Mieke Jansen |
| E-mail(s) |
Mieke.Jansen@bio.kuleuven.be
|
| Organization name |
KULeuven
|
| Department |
Biology
|
| Lab |
Laboratory of Aquatic Biology and Evolutionary Ecology
|
| Street address |
Ch. Deberiotstraat 32
|
| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL10966 |
| Series (1) |
| GSE24322 |
Stressor mRNA expression profiling of three different stressors in the water flea Daphnia magna |
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