|
| Status |
Public on Jan 03, 2008 |
| Title |
Includes Samples from control group pool C2 at T4 (Hyb15Cy5) |
| Sample type |
RNA |
| |
|
| Source Name |
hemocytes from Penaeus monodon exposed to environmental stressors
|
| Organism(s) |
Penaeus monodon |
| Characteristics |
Samples used in this experiment came from hemocytes of juvenile Penaeus monodon originating from a hatchery in North Queensland, Australia. This hatchery uses wild caught broodstock for its operation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from haemocytes by homogenisation in Trizol-LS(Life Technologies) and then amplified using the Message Amp II® aRNA amplification kit (Ambion®) following the manufacturer’s instructions for one-round amplification.
|
| Label |
Cy5
|
| Label protocol |
cDNA labeling was performed as previously described (Woods et al. 2004, Development 131: 2921-2933). Amplified RNA (2μg) were reverse transcribed using an oligo dT18 primer (50 μM), 1.25 mM dNTP mix (Biotech International) with a ratio of 4:1 aminoallyl-dUTP to dTTP (Sigma), 10 mM dithiothreitol (DTT), 600 units of Superscript III ® (Invitrogen), and 60 units of RNAse out (Invitrogen) in a 30 μl reaction volume. RNA was hydrolyzed by the addition of 3 μl of 1 M NaOH and 3 μl of 0.5 M ethylene-diamine-tetraacetate (EDTA) followed by heating to 65°C for 15 min. The solution was neutralized by the addition of 3 μl of 1 M HCl. Following synthesis, aminoallyllabeled cDNA (aa-cDNA) was purified using the Millipore Montage PCR cleanup kit following the manufacturer’s instructions, dried down and the pellet resuspended in 4.5 μl of freshly prepared 100 mM sodium carbonate (pH 9.0). A total of 100 ng of Cy3 or Cy5 reactive dyes (resuspended in 4.5 μl DMSO) (Amersham) were added to the respective aa-cDNAs and the coupling reaction allowed to proceed at room temperature for 1.5 h in the dark. Unincorporated dyes were removed from the reaction by the addition of 4 M hydroxylamine. Cy3- and Cy5-labelled cDNA was then combined and purified using a Qiagen QIAquick PCR cleanup kit
|
| |
|
| Hybridization protocol |
Human Cot1 DNA [(10 μg), (Invitrogen)], and 2 μg of poly dA (Roche) were added to the purified probe mix and dried prior to resuspending in 30 μl of 4X SSC (0.6 M sodium chloride and 60 mM sodium citrate), 40% deionized formamide and 0.1% sodium dodecyl sulfate (SDS). Following incubation at 95°C for 5 min, and then 45°C for 90 min, the labelled probe mix was loaded onto the microarray chip, and hybridisation was carried out for 16 h at 45°C. Following hybridisation, the microarray chip was washed twice with 0.2X SSC, 0.05% SDS and 2 more times with 0.2X SSC. Slides were finally dried by centrifugation at 600 rpm for 5 min.
|
| Scan protocol |
Cy3 and Cy5 fluorescence hybridized to the cDNA elements spotted onto the microarray chip was detected with a GMS 418 Array Scanner (Genetic Microsystems) using Imagene 4.2 software (BioDiscovery, Inc.) and the default parameters.
|
| Description |
includes samples from control group pool C2 at T4
|
| Data processing |
Only genes with a signal to noise ratio (computed by dividing the backgroundcorrected intensities by the standard deviation of the background pixels) greater than 1.0, scored in both dyes and with at least 24 readings across the 24 slides were used in this study. The VALUE data included in this table was calculated by base-2 logarithm transforming (Log2) the value obtained by subtracting signal_median - background_median. Signal intensities were base-2 logarithm (log2) transformed and normalized by a multivariate mixed-model approach as previously described (Reverter et. al. 2005. Bioinformatics 21: 1112-1120) using the following equation: Only genes with a signal to noise ratio (computed by dividing the backgroundcorrected intensities by the standard deviation of the background pixels) greater than 1.0, scored in both dyes and with at least 24 readings across the 24 slides were used in this study. Signal intensities were base-2 logarithm (log2) transformed and normalized by a multivariate mixed-model approach as previously described (Reverter et. al. 2005. Bioinformatics 21: 1112-1120) using the following equation. yE = XEßE + ZE1cE + ZE2aE + ZE3dE + ZE4tE + eE Where: y is the vector of all the intensity signals (N= 202 636), background corrected and base-2 log transformed X is the incidence matrix relating signals in y with systematic fixed effects in ß including array slide, printing block and fluorescent dye channel ZE1 is the incidence matrix relating y with random effects in c corresponding to the clones (N= 2841) printed on the array that passed the filtering criteria ZE2 is the incidence matrix relating y with random effects in a corresponding to the three-way interaction of clone by array and printing block ZE3 is the incidence matrix relating y with random effects in d corresponding to the interaction of clone by the two fluorescent dye channels ZE4 is the incidence matrix relating y with random effects in t corresponding to the interaction of clone by the 12 experimental treatment conditions e is the random error associated with signals in y. The variance components were estimated by restricted maximum likelihood (REML) using VCE 4 software (available at Eildert Groeneveld's web page at the Institute for Animal Breeding Mariensee). The fitting of the above model provided BLUP (Best Linear Unbiased Prediction) solutions that were the basis for the normalization methods.
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| |
|
| Submission date |
Apr 02, 2007 |
| Contact name |
Enrique de la Vega |
| E-mail(s) |
delavega@musc.edu
|
| Phone |
1-843-7628820
|
| Fax |
1-843-7628737
|
| Organization name |
Medical University of South Carolina
|
| Department |
Biochemistry
|
| Lab |
Hollings Marine Laboratory
|
| Street address |
331 Fort Johnson Road
|
| City |
Charleston |
| State/province |
SC |
| ZIP/Postal code |
29412 |
| Country |
USA |
| |
|
| Platform ID |
GPL4890 |
| Series (1) |
| GSE7456 |
Stress induced gene expression profiling in Penaeus monodon |
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