|
| Status |
Public on Apr 08, 2013 |
| Title |
Colony 3-2 (cy5) - Colony 1-2 (cy3) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Colony 3, within-colony biological replicate 2
|
| Organism |
Acropora millepora |
| Characteristics |
genotype/variation: intron 4-500 haplotype 1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Coral fragments were immediately frozen in liquid nitrogen upon sampling. Frozen coral fragments were homogenized in Trizol Reagent (Invitrogen). Trizol RNA extraction protocol was followed as per manufacturer’s protocol through phase separation, after which the aqueous phase was recovered, mixed with an equal volume of absolute ethanol, and processed with an RNeasy Mini kit (QIAGEN). RNA integrity was assessed with the use of a Bioanalyzer (Agilent); only high integrity RNA was used for cDNA synthesis and subsequent hybridization.
|
| Label |
Cy5
|
| Label protocol |
cDNA probe synthesis was performed from 2.5 μg high-quality total RNA using Superscript II Reverse Transcriptase (Invitrogen) and the 3DNA Array 350 kit (Genisphere) according to the manufacturer’s guidelines. Two primers were used, which consisted of a Cy3 and Cy5 specific capture sequence and a poly-T tail. In order to make each sample specific for Cy3 and Cy5, each sample was reverse transcribed separately using only one primer.
|
| |
|
| Channel 2 |
| Source name |
Colony 1, within-colony biological replicate 2
|
| Organism |
Acropora millepora |
| Characteristics |
genotype/variation: intron 4-500 haplotype 1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Coral fragments were immediately frozen in liquid nitrogen upon sampling. Frozen coral fragments were homogenized in Trizol Reagent (Invitrogen). Trizol RNA extraction protocol was followed as per manufacturer’s protocol through phase separation, after which the aqueous phase was recovered, mixed with an equal volume of absolute ethanol, and processed with an RNeasy Mini kit (QIAGEN). RNA integrity was assessed with the use of a Bioanalyzer (Agilent); only high integrity RNA was used for cDNA synthesis and subsequent hybridization.
|
| Label |
Cy3
|
| Label protocol |
cDNA probe synthesis was performed from 2.5 μg high-quality total RNA using Superscript II Reverse Transcriptase (Invitrogen) and the 3DNA Array 350 kit (Genisphere) according to the manufacturer’s guidelines. Two primers were used, which consisted of a Cy3 and Cy5 specific capture sequence and a poly-T tail. In order to make each sample specific for Cy3 and Cy5, each sample was reverse transcribed separately using only one primer.
|
| |
|
| |
| Hybridization protocol |
Static hybridizations were performed using formamide-based hybribization buffer (Genisphere). Prehybridization used 1 μg Human Cot-1 DNA for 90 minutes. Hybridization with cDNA was performed for 14 hours at 47°C. Arrays were washed in 65°C 2x SSC/0.2% SDS for 15 minutes, 2x SSC at room temperature for 15 minutes, and 0.2x SSC at room temperature for 15 minutes. Dye capture with Array 900 3DNA capture reagents (Genisphere) was performed at 50°C for 4 hours, followed by washing. Arrays were dipped in DyeSaver II (Genisphere).
|
| Scan protocol |
Scanning was performed on a GenePix Personal 4100A (Axon Instruments) microarray scanner using GenePix Pro 4.1.
|
| Data processing |
Following gridding and quality control using GenePix Pro 4.1 (Axon Instruments), background-subtracted mean intensity values were log- and lowess-transformed using R/Maanova version 1.18 (Wu and Churchill 2008). A fixed-effect ANOVA model was fit to the normalized data. Empirical-Bayes Fs statistic was used to test for differentially expressed genes at each sampling time. P-values for each clone were calculated from 500 permutations of residual shuffling. False discovery rate adjustments were implemented using jsFDR (Storey 2002, J Roy Stat Soc B), using an adjusted p-value threshold of less than 0.05.
|
| |
|
| Submission date |
Dec 03, 2012 |
| Last update date |
Apr 08, 2013 |
| Contact name |
Anthony John Bellantuono |
| Organization name |
Florida International University
|
| Department |
Biological Sciences
|
| Lab |
Rodriguez-Lanetty
|
| Street address |
11200 SW 8th
|
| City |
Miami |
| State/province |
FL |
| ZIP/Postal code |
33199 |
| Country |
USA |
| |
|
| Platform ID |
GPL6941 |
| Series (1) |
| GSE42684 |
High natural gene expression variation in the reef-building coral Acropora millepora: Potential for acclimative and adaptive plasticity |
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