|
| Status |
Public on May 06, 2009 |
| Title |
Adult-Polyp2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
polyp pool 2
|
| Organism |
Orbicella faveolata |
| Characteristics |
developmental stage: polyp
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from frozen coral tissues was isolated using Qiazol lysis reagent (Qiagen) following product specifications. Both adult and larval tissues were homogenized using a pre-chilled mortar and pestle. Frozen coral powder was transferred directly to Qiazol. Two chloroform extractions were performed, followed by isopropanol precipitation and two washes in 80% ethanol. RNA pellets were re-dissolved in nuclease-free water and cleaned further with RNeasy Mini columns (Qiagen). RNA quantity and integrity were assessed with a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. All RNA samples were amplified using the MessageAmp II aRNA kit (Ambion) with 1 µg of total RNA as input.
|
| Label |
Cy3, Cy5
|
| Label protocol |
Five µg of aRNA per sample were primed with 5 µg/µL random nonamer for 10 min at 70oC. Reverse transcription (RT) lasted for 2 hr at 50oC using a master mix containing a 4:1 ratio of aminoallyl-dUTP to TTP. Following RT, single-stranded RNA was hydrolyzed by incubating the RT reactions in 10 µL 0.5 M EDTA and 10 µL 1 M NaOH for 15 min at 65oC. After hydrolysis, RT reactions were cleaned using MinElute Reaction Purification columns (Qiagen). Cy3 and Cy5 dyes (GE Healthcare) were dissolved in 12 µL DMSO, and the coupling reactions lasted for 2 hr at room temperature in the dark.
|
| |
|
| Channel 2 |
| Source name |
adult fragment 2
|
| Organism |
Orbicella faveolata |
| Characteristics |
developmental stage: adult
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from frozen coral tissues was isolated using Qiazol lysis reagent (Qiagen) following product specifications. Both adult and larval tissues were homogenized using a pre-chilled mortar and pestle. Frozen coral powder was transferred directly to Qiazol. Two chloroform extractions were performed, followed by isopropanol precipitation and two washes in 80% ethanol. RNA pellets were re-dissolved in nuclease-free water and cleaned further with RNeasy Mini columns (Qiagen). RNA quantity and integrity were assessed with a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. All RNA samples were amplified using the MessageAmp II aRNA kit (Ambion) with 1 µg of total RNA as input.
|
| Label |
Cy3, Cy5
|
| Label protocol |
Five µg of aRNA per sample were primed with 5 µg/µL random nonamer for 10 min at 70oC. Reverse transcription (RT) lasted for 2 hr at 50oC using a master mix containing a 4:1 ratio of aminoallyl-dUTP to TTP. Following RT, single-stranded RNA was hydrolyzed by incubating the RT reactions in 10 µL 0.5 M EDTA and 10 µL 1 M NaOH for 15 min at 65oC. After hydrolysis, RT reactions were cleaned using MinElute Reaction Purification columns (Qiagen). Cy3 and Cy5 dyes (GE Healthcare) were dissolved in 12 µL DMSO, and the coupling reactions lasted for 2 hr at room temperature in the dark.
|
| |
|
| |
| Hybridization protocol |
Dye-coupled cDNAs were cleaned (MinElute columns), and appropriate Cy3 and Cy5 labeled cDNAs were mixed together in a hybridization buffer containing 0.25 % SDS, 25 mM HEPES, and 3x SSC. The hybridization mixtures were boiled for 2 min at 99oC then allowed to cool at room temperature for 5 min. The cooled hybridization mixtures were pipetted under a mSeries Lifterslip (Erie Scientific), and hybridization took place in Corning hybridization chambers overnight at 63oC. Microarrays were washed twice in 0.6x SSC and 0.01 % SDS followed by a rinse in 0.06x SSC and dried via centrifugation.
|
| Scan protocol |
All microarrays were scanned using an Axon 4000B scanner (Molecular Devices) where care was taken to balance photomultiplier tube (PMT) settings.
|
| Description |
n/a
n/a
|
| Data processing |
Microarray data analysis -- The experiment followed a loop design in which all life stages (i.e. planula, polyp, adult) were directly compared against each other. Hybridizations were performed in triplicate (n=3 for each developmental stage) and included dye swapping between technical replicates. Pre-processing of microarray intensity data was performed using TIGR TM4 software. Briefly, spot-finding was performed in SpotFinder 3.11 with background-subtracted median intensities being extracted. Using MIDAS 2.19, the data were first LOWESS normalized, and the dye swap pairs were collapsed; and secondly, the in-slide duplicates were collapsed. Resulting intensities in both channels (for nine total microarrays) were compiled into a spreadsheet. Genes were included in subsequent statistical analysis only if there were data in two out of three replicates for each developmental stage. Of 1310 possible genes, 1243 passed filtering criteria. The ratio between the fluorescence intensity of the two channels was used as input for BAGEL (Bayesian Analysis of Gene Expression Levels). The BAGEL software uses Bayesian probability to infer a relative expression level of each gene. An estimated mean and 95% credible interval of the relative level of expression of each gene is computed in each treatment and time point. We used the conservative gene-by-gene criterion of non-overlapping 95% credible intervals to regard a gene as differentially expressed.
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| |
|
| Submission date |
May 05, 2009 |
| Last update date |
May 05, 2009 |
| Contact name |
Michael DeSalvo |
| E-mail(s) |
[email protected]
|
| Organization name |
UC San Francisco
|
| Department |
Department of Anesthesia
|
| Lab |
Roland Bainton
|
| Street address |
600 16 St Box 2200
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158-2200 |
| Country |
USA |
| |
|
| Platform ID |
GPL6515 |
| Series (1) |
| GSE15962 |
Gene expression analysis during metamorphosis and the onset of calcification in the scleractinian coral M. faveolata |
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