|
| Status |
Public on Apr 01, 2009 |
| Title |
mf late con2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
reference
|
| Organism |
Orbicella faveolata |
| Characteristics |
developmental stage: larva
other: reference
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of coral larvae was isolated using Qiazol (Qiagen) according to manufacturer’s instructions. Larvae were homogenized for 2 minutes using a Mini-Beadbeater (Biospec) with 0.1 mm and 0.55 mm silica beads to disrupt cellular structures. RNA pellets were cleaned further with RNeasy Mini columns (Qiagen).
|
| Label |
cy3
|
| Label protocol |
1 µg of total RNA was amplified using the MessageAmp II aRNA kit (Ambion). For cDNA synthesis, 3 µg of aRNA per sample were primed with 3.5 nmol random pentadecamer for 10min at 70°C. Reverse transcription (RT) lasted for 2 hours at 42°C using a master mix containing a 3:2 ratio of aminoallyl-dUTP to TTP. Following RT, single-stranded RNA was hydrolyzed by incubating the RT reactions in 10 uL 0.5M EDTA and 10 uL 1M NaOH for 15min at 65°C. After hydrolysis, RT reactions were cleaned using the MinElute Cleanup kit (Qiagen). Cy3 and Cy5 dyes (GE Healthcare) were dissolved in 17uL DMSO, and the coupling reactions lasted for 2hr at room temperature in the dark. Dye-coupled cDNAs were cleaned using the MinElute Cleanup kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
mf late con2
|
| Organism |
Orbicella faveolata |
| Characteristics |
developmental stage: larva
other: sample
protocol: untreated
time: late
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of coral larvae was isolated using Qiazol (Qiagen) according to manufacturer’s instructions. Larvae were homogenized for 2 minutes using a Mini-Beadbeater (Biospec) with 0.1 mm and 0.55 mm silica beads to disrupt cellular structures. RNA pellets were cleaned further with RNeasy Mini columns (Qiagen).
|
| Label |
cy5
|
| Label protocol |
1 µg of total RNA was amplified using the MessageAmp II aRNA kit (Ambion). For cDNA synthesis, 3 µg of aRNA per sample were primed with 3.5 nmol random pentadecamer for 10min at 70°C. Reverse transcription (RT) lasted for 2 hours at 42°C using a master mix containing a 3:2 ratio of aminoallyl-dUTP to TTP. Following RT, single-stranded RNA was hydrolyzed by incubating the RT reactions in 10 uL 0.5M EDTA and 10 uL 1M NaOH for 15min at 65°C. After hydrolysis, RT reactions were cleaned using the MinElute Cleanup kit (Qiagen). Cy3 and Cy5 dyes (GE Healthcare) were dissolved in 17uL DMSO, and the coupling reactions lasted for 2hr at room temperature in the dark. Dye-coupled cDNAs were cleaned using the MinElute Cleanup kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
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 99°C then allowed to cool at room temperature for 5 minutes. The cooled hybridization mixtures were pipetted under an mSeries Lifterslip (Erie Scientific), and hybridization took place in Corning hybridization chambers overnight at 63°C. 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 |
Slides were immediately scanned using an Axon 4000B scanner.
|
| Description |
Pooled reference made out of equal amounts of RNA from all samples
Pooled untreated control larvae late
|
| Data processing |
Spot intensities were extracted, background subtracted, and block-wise lowess normalized using TIGR Spotfinder 2.2.4 (Saeed et al., 2003). Duplicated spots were averaged over the median of intensities. Only genes that were called in 2 out of 3 arrays for every experimental condition were subjected to further analysis.
|
| |
|
| Submission date |
Feb 20, 2009 |
| Last update date |
Mar 02, 2009 |
| Contact name |
Christian Robert Voolstra |
| E-mail(s) |
[email protected]
|
| URL |
http://faculty.kaust.edu.sa/sites/christianvoolstra/Pages/home.aspx
|
| Organization name |
King Abdullah University of Science and Technology
|
| Street address |
Red Sea Research Center
|
| City |
Thuwal |
| ZIP/Postal code |
23955 |
| Country |
Saudi Arabia |
| |
|
| Platform ID |
GPL8215 |
| Series (1) |
| GSE14923 |
The host transcriptome remains unaltered during the establishment of coral-algal symbioses |
|
