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Sample GSM599769 Query DataSets for GSM599769
Status Public on Sep 01, 2013
Title ref_fish_144_1
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 fish exposed, 144h, r1
Organism Daphnia magna
Characteristics clone: P 32,85
gender: female
treatment: fish exposure
time point: 144h
replicate: 1
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 17_300309
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

Data table header descriptions
ID_REF
VALUE Average log2 Lowess-normalized ratio FC (Cy5/Cy3)

Data table
ID_REF VALUE
EBT_DM_CarbU_P14A1 -0.018460409
EBT_DM_CarbU_P14A10 0.228370846
EBT_DM_CarbU_P14A11 -0.259847277
EBT_DM_CarbU_P14A12 0.183994669
EBT_DM_CarbU_P14A2 -0.232659628
EBT_DM_CarbU_P14A3 0.261109273
EBT_DM_CarbU_P14A4 0.124140229
EBT_DM_CarbU_P14A5 0.03444209
EBT_DM_CarbU_P14A6 0.021754796
EBT_DM_CarbU_P14A7 -0.243461428
EBT_DM_CarbU_P14A8 -0.211861573
EBT_DM_CarbU_P14A9 0.008985075
EBT_DM_CarbU_P14B1 -0.156535425
EBT_DM_CarbU_P14B10 0.157502837
EBT_DM_CarbU_P14B11 0.170457843
EBT_DM_CarbU_P14B12 0.209651167
EBT_DM_CarbU_P14B2 0.121887099
EBT_DM_CarbU_P14B3 0.250185613
EBT_DM_CarbU_P14B4 -0.243017508
EBT_DM_CarbU_P14B5 -0.168208924

Total number of rows: 1918

Table truncated, full table size 55 Kbytes.




Supplementary file Size Download File type/resource
GSM599769.gpr.gz 578.1 Kb (ftp)(http) GPR
Processed data included within Sample table
Processed data are available on Series record

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