VALUE = Lowess-normalized log2 (Cy5/Cy3) ratio
Dye-coupled cDNAs were cleaned (MinElute), and 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 5min. The cooled hybridization mixtures were pipetted under an mSeries Lifterslip (Erie Scientific), and hybridization took place in custom-fabricated 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.
One μg of total RNA was amplified using the MessageAmp II aRNA kit (Ambion), and 3 μg of aRNA per sample were primed with 10 μg 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 and SuperScript III reverse transcriptase (Invitrogen). Following RT, single-stranded RNA was hydrolyzed by incubating the RT reactions in 10 μL 0.5M EDTA and 10 μL 1M sodium hydroxide for 15 min at 65oC. After hydrolysis, RT reactions were cleaned using MinElute Reaction Cleanup columns (Qiagen). Cy3 and Cy5 dyes (GE Healthcare) were dissolved in 18 μL dimethyl sulfoxide, and the coupling reactions lasted for 1 hr at room temperature in the dark.
Gridding was performed using GenePix Pro 6.0. TIGR Express Converter was used to convert GenePix results files into .mev files for input into TIGR MIDAS 2.19 (Saeed et al. 2003). Data for a particular spot were discarded according to the stringent one bad channel policy. Additional data were discarded if spots had been flagged during gridding in GenePix, and if the intensity in either channel was less than 50,000 units. After filtering, background-subtracted median intensity values were LOWESS normalized, and in-slide duplicates were averaged.
Slides were immediately scanned using an Axon 4000B scanner (Molecular Devices), where care was taken to balance photomultiplier tube (PMT) settings.
All aquaria were exposed to shaded ambient light during a four day acclimation period, and these conditions remained for the control aquaria during the entire experiment (roughly 40% of that experienced in the natural reef setting). Temperature in all aquaria during acclimation and experimental periods differed slightly. Following the acclimation period, a fragment from each control and experimental aquaria was sampled (t0-C and t0-D). After time zero sampling, the fiberglass pond containing the experimental tanks was covered with black plastic. The light exposure in the experimental tanks following time zero sampling was 0% of that experienced in the natural reef setting. After 9 days of darkness stress, a fragment of A. palmata was sampled from each control and experimental aquaria (9d-C and 9d-D). All samples were taken at night and frozen in liquid nitrogen.
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. Frozen coral powder was transferred directly to Qiazol. Three chloroform extractions were performed, followed by isopropanol precipitation and three washes in 70% 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.