|
| Status |
Public on May 31, 2008 |
| Title |
Mfav_bleaching_rep1_dyeswap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source Name |
control fragment 1
|
| Organism(s) |
Montastraea faveolata |
| Characteristics |
fragment kept at control temperature for 11 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiazol used to extract total RNA.
RNeasy Mini kit used to clean RNA.
Details: 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 80% 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. Microarray protocols followed those established by the Center for Advanced Technology at the University of California, San Francisco (http://cat.ucsf.edu/).
|
| Label |
Cy5
|
| Label protocol |
Labeled using Amersham CyDye Post-labeling kit.
http://cat.ucsf.edu/pdfs/hybCourseProtocols_v2.7.pdf
Specifically, 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 42oC 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 10uL 0.5M EDTA and 10uL 1M NaOH for 15min at 65oC. After hydrolysis, RT reactions were cleaned using Zymo Clean and Concentrator 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 |
bleached fragment 3
|
| Organism(s) |
Montastraea faveolata |
| Characteristics |
fragment thermal stressed for 11 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiazol used to extract total RNA.
RNeasy Mini kit used to clean RNA.
Details same as for channel 1
|
| Label |
Cy3
|
| Label protocol |
Labeled using Amersham CyDye Post-labeling kit.
http://cat.ucsf.edu/pdfs/hybCourseProtocols_v2.7.pdf
Details same as for channel 1
|
| |
|
| |
| 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 (Zymo), 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 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. Slides were immediately scanned using an Axon 4000B scanner. Microarray data analysis followed a dye-swap design where each of the 5 control fragments was randomly paired to one of the 5 heat-stressed fragments. Each hybridization was performed twice with dye swapping between technical replicates, which allows for the measurement of dye labeling bias.
http://cat.ucsf.edu/pdfs/hybCourseProtocols_v2.7.pdf
|
| Scan protocol |
Scanned using Axon 4100B scanner with GenePix 6.0 software.
|
| Description |
A single time point, single genotype thermal stress experiment was performed. A large colony of Montastraea faveolata was divided into ten coral “plugs” using a 2.5cm diameter punch tool. The fragments were divided evenly between 2 identical 75-liter aquaria, ca. 25cm deep, and kept at ambient temperature (29.23±0.48oC) for 3 days. After the acclimation period, 2 200-Watt aquarium heaters were turned on in the experimental aquarium, which subsequently heated to 32.23±0.48oC. The experiment lasted 10 days and 17 hours, after which all fragments were frozen in liquid nitrogen for molecular analysis.
|
| Data processing |
Data analysis was performed using J/MAANOVA in the R statistical environment. Background-subtracted mean intensity values were log2 transformed and normalized using the spatial-intensity joint lowess algorithm. A fixed-effect MAANOVA model was fit to the intensity data with array, sample, and dye terms. Differentially expressed genes were identified by testing the sample term according to the Empirical-Bayes Fs statistic. P-values for each gene were generated by shuffling the residuals over 500 permutations. FDR adjustments were applied to the p-values using a step-down FDR control method.
|
| |
|
| Submission date |
Feb 25, 2008 |
| Contact name |
Michael DeSalvo |
| E-mail(s) |
mdesalvo@ucmerced.edu
|
| Organization name |
UC Merced
|
| Department |
School of Natural Sciences
|
| Lab |
Monica Medina
|
| Street address |
P.O. Box 2039
|
| City |
Merced |
| State/province |
CA |
| ZIP/Postal code |
95344 |
| Country |
USA |
| |
|
| Platform ID |
GPL6515 |
| Series (2) |
| GSE10630 |
Mfaveolata single-time point bleaching experiment |
| GSE10680 |
Montastraea faveolata bleaching study |
|
