clone: P 32,85 gender: female treatment: control time point: 96h 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
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.
clone: P 32,85 gender: female treatment: fish exposure time point: 96h 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
46_020409 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).
