Total RNA Qiazol extraction, aRNA amplification, cDNA Superscript III
Label
cy3 (IA)
Label protocol
Total RNA of approximately 1,000 coral embryos was isolated using Qiazol lysis reagent (Qiagen) according to manufacturer's instructions. RNA quantity and integrity was assessed using a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. For each sample, 1ug of total RNA was amplified using the MessageAmp II aRNA kit (Ambion). Subsequent cDNA synthesis and dye-labeling was performed using 3 ug 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). Appropriate Cy3 and Cy5 labeled cDNAs were mixed together in a hybridization buffer containing 0.25% SDS, 25 mM HEPES and 3 × SSC, resulting in a final volume of 70ul.
Total RNA Qiazol extraction, aRNA amplification, cDNA Superscript III
Label
cy3 (IB)
Label protocol
Total RNA of approximately 1,000 coral embryos was isolated using Qiazol lysis reagent (Qiagen) according to manufacturer's instructions. RNA quantity and integrity was assessed using a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. For each sample, 1ug of total RNA was amplified using the MessageAmp II aRNA kit (Ambion). Subsequent cDNA synthesis and dye-labeling was performed using 3 ug 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). Appropriate Cy3 and Cy5 labeled cDNAs were mixed together in a hybridization buffer containing 0.25% SDS, 25 mM HEPES and 3 × SSC, resulting in a final volume of 70ul.
Hybridization protocol
Appropriate Cy3 and Cy5 labeled cDNAs were mixed together in a hybridization buffer containing 0.25% SDS, 25 mM HEPES and 3 × SSC, resulting in a final volume of 70μl. The hybridization mixtures were boiled for 2 min at 99°C and allowed to cool at room temperature for 5 min. 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 0.6 × SSC, 0.01% SDS and were kept in 0.06 × SSC until analysis.
Scan protocol
Slides were dried via centrifugation and scanned using an Axon 4000B scanner. The experimental setup followed a reference design, i.e. all samples were hybridized against the same pool made up of equal amounts of aRNA from all samples.
Data processing
TIGR Spotfinder 2.2.3 was used to extract spot intensities and subtract background (Saeed et al. 2003). The data were normalized using TIGR MIDAS 2.21 printtip-specific LOWESS followed by in-slide replicates analyses (Saeed et al. 2003).
