Each sample contaiend 50 mg gill from three individuals of each family. Total RNA were extracted using the Qiagen miniprep system and on-column DNAse treatment. Total RNA were used as template for synthesizing antisense RNA using the Amino Allyl MessageAmp™ Kit (Ambion, Inc., Austin, TX). Each reaction included RNA standards: 100 ng chlorophyll A/B binding protein (Genbank #CO059871) RNA, 10 ng photosystem core protein (Genbank #CO062297) RNA, 1 ng flavodoxin (Genbank #CO065421) RNA, and 0.1 ng photolyase (Genbank #CO064781) RNA.
Label
Cy3
Label protocol
1 microgram of antisense RNA was labeled with Cy3 dye according to the protocol for the Amino Allyl MessageAmp™ Kit (Ambion, Inc., Austin, TX).
Hybridization protocol
Each slide hybridized with 90-μl mixture containing 20 μg of Cy3-coupled aRNA in hybridization buffer [50% formamide, 2.4% SDS, 4xSSPE, 2.5x Denhardt’s solution, plus 1 µg Cot-DNA and 1 µg polydATP]. Hybridizations were conducted at 50°C in a humidified hybridization oven (Boekel Inslide-Outtm, Boekel, Festerville, PA) for 12 h.
Scan protocol
ScanArray Express (Perkin Elmer, Boston, MA) microarray scanner, 70% PMT gain and 90% laser power.The QuantArray software package (Perkin Elmer, Boston, MA) was used to acquire raw fluorescence data, background, and spot quality information from the scanned images using the included Histogram spot segmentation method. Each spot image was visually inspected for overall quality, and damaged spots were excluded prior to statistical analysis.
Description
Family 6 Time 0 Replicate 2
Data processing
We transformed the entire dataset using a variance stabilization normalization method (vsn; Huber et al. 2002). All spots were included in the transformation. Spot pairs were excluded whose average intensity across the 60 slides was less than 6 for at least 1 of the sampling times, and we excluded all unsequenced spots. We omitted flagged spots prior to statistical analysis.
