|
| Status |
Public on May 31, 2009 |
| Title |
Recovered1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Mfav_CBR_pooled_reference
|
| Organism |
Orbicella faveolata |
| Characteristics |
sample: Pool of all control samples included in the experiment
|
| Treatment protocol |
On the night of 1 September, control time point samples from five different colonies were collected from each tank (Figure 1), before one 200-Watt aquarium heater was turned on in the treatment aquarium. A second heater was turned on 3 days later. During the thermal stress experiment, the control aquarium received mean water temperature of 28.8 ± 1.2oC; the heated aquarium, 31.5 ± 1.1oC. During the thermal stress experiment, tanks received mean PAR of 420 ± 152 µmol m-2 s-1. On 11 September, and 19 October, one sample each from five different colonies was collected from each tank for the bleaching and recovery time point, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from all frozen coral fragments was isolated using Qiazol lysis reagent (QIAGEN). Live tissue was chiseled off each coral fragment and homogenized using a pre-chilled mortar and pestle embedded in dry ice. Frozen coral powder was transferred directly to Qiazol. Two chloroform extractions were performed, followed by isopropanol precipitation and 2 washes in 70% ethanol. RNA pellets were re-dissolved in nuclease-free water and cleaned further over RNeasy Mini columns (QIAGEN). RNA quality and quantity were assessed with a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
1ug of total RNA was amplified using the MessageAmp II aRNA kit (Ambion), and 3ug of aRNA per sample were primed with 5ug/uL random nonamer for 10min at 70oC. Reverse transcription (RT) lasted for 2hr 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 10uL 0.5M EDTA and 10uL 1M NaOH for 15min at 65oC. After hydrolysis, RT reactions were cleaned using Qiagen MinElute reaction purification columns. Cy3 and Cy5 dyes (GE Healthcare) were dissolved in 17uL DMSO, and the coupling reactions lasted for 2hr at room temperature in the dark.
|
| |
|
| Channel 2 |
| Source name |
Recovered1
|
| Organism |
Orbicella faveolata |
| Characteristics |
sample: Fragment that had regained symbionts following experimental bleaching
|
| Treatment protocol |
On the night of 1 September, control time point samples from five different colonies were collected from each tank (Figure 1), before one 200-Watt aquarium heater was turned on in the treatment aquarium. A second heater was turned on 3 days later. During the thermal stress experiment, the control aquarium received mean water temperature of 28.8 ± 1.2oC; the heated aquarium, 31.5 ± 1.1oC. During the thermal stress experiment, tanks received mean PAR of 420 ± 152 µmol m-2 s-1. On 11 September, and 19 October, one sample each from five different colonies was collected from each tank for the bleaching and recovery time point, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from all frozen coral fragments was isolated using Qiazol lysis reagent (QIAGEN). Live tissue was chiseled off each coral fragment and homogenized using a pre-chilled mortar and pestle embedded in dry ice. Frozen coral powder was transferred directly to Qiazol. Two chloroform extractions were performed, followed by isopropanol precipitation and 2 washes in 70% ethanol. RNA pellets were re-dissolved in nuclease-free water and cleaned further over RNeasy Mini columns (QIAGEN). RNA quality and quantity were assessed with a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
1ug of total RNA was amplified using the MessageAmp II aRNA kit (Ambion), and 3ug of aRNA per sample were primed with 5ug/uL random nonamer for 10min at 70oC. Reverse transcription (RT) lasted for 2hr 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 10uL 0.5M EDTA and 10uL 1M NaOH for 15min at 65oC. After hydrolysis, RT reactions were cleaned using Qiagen MinElute reaction purification columns. Cy3 and Cy5 dyes (GE Healthcare) were dissolved in 17uL DMSO, and the coupling reactions lasted for 2hr at room temperature in the dark.
|
| |
|
| |
| Hybridization protocol |
Prior to hybridization, microarrays were post-processed by: 1) UV crosslinking at 60 mJ; 2) a “shampoo” treatment (3x SSC, 0.2% SDS at 65oC); 3) blocking with 5.5g succinic anhydride dissolved in 335mL 1-methyl-2-pyrrilidinone and 15mL sodium borate; and 4) drying via centrifugation. Dye-coupled cDNAs were cleaned (Qiagen MinElute), and appropriate Cy3 and Cy5 labeled cDNAs were mixed together in a hybridization buffer containing 0.25% SDS, 25mM HEPES, and 3x SSC. The hybridization mixtures were boiled for 2min at 99oC then allowed to cool at room temperature for 5min. The cooled hybridization mixtures were pipetted under an mSeries Lifterslip (Erie Scientific), and hybridization took place 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
|
| Data processing |
TIFF images were generated with GenePix Pro 6.0, and gridding was performed using TIGR Spotfinder 3.1.1 with the Otsu segmentation method and background correction (the top 25% of background pixels were discarded prior to local background estimation). Using TIGR MIDAS 2.19, background-corrected data were LOWESS normalized, and in-slide duplicates were averaged. Genes were included in statistical analyses only if there were data for three out of five hybridizations for a given category (i.e., control, bleaching, or recovery).
|
| |
|
| Submission date |
Mar 16, 2009 |
| Last update date |
Mar 17, 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 |
GPL7317 |
| Series (2) |
| GSE15253 |
Symbiodinium clade content drives host transcriptome more than thermal stress in the coral Montastraea faveolata part 2 |
| GSE15262 |
Symbiodinium clade content drives host transcriptome more than thermal stress in the coral Montastraea faveolata |
|
