GRASP microarray studies were conducted using liver samples. To minimize technical variability within a study, all targets were synthesized in one round, and all hybridizations were conducted simultaneously on slides from a single batch (EB017). Total RNA, prepared from flash-frozen adult liver tissues using TRIzol reagent and methods (Invitrogen), was quantified and quality-checked by spectrophotometer and agarose gel. The microarray experiments used pooled RNA templates: pooled C contained equal quantities from 10 individual C fish, pooled R contained equal quantities from 10 individual R fish, and pooled T contained equal quantities from 10 individual T fish. Microarray experimental design involved 3 comparisons (C vs. R, C vs. T, R vs. T), each run in duplicate (1 chip and a dye flip). Microarray hybridizations were performed using the Genisphere Array50™ kit and instructions. Briefly, 20 ug total RNA (pooled C, pooled R, or pooled T) were reverse transcribed using oligo d(T) primers with 3’ or 5’ unique sequence overhangs for the Cy3 or Cy5 labeling reactions, respectively. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95˚C MilliQ H2O, and drying by centrifugation (5 min 2000 rpm in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 48˚C water bath. The coho salmon liver cDNAs were hybridized to the salmonid cDNA microarray in a formamide-based buffer (25% formamide, 4X SSC, 0.5% SDS, 2X Denhardt's solution) 16 h at 48˚C. Coverslips were floated off in 48˚C (2X SSC, 0.1% SDS), and arrays were washed 1 X 10 min in 2X SSC, 0.1% SDS at 48˚C, 2 X 5 min in 2X SSC, 0.1% SDS at room temperature (RT), 2 X 5 min in 1X SSC at RT, and 2 X 5 min in 0.1X SSC at RT, and dried by centrifugation as before. The Cy3 and Cy5 3-dimensional fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized to the bound cDNA on the microarray in a formamide-based buffer (25% formamide, 4X SSC, 0.5% SDS, 2X Denhardt's solution) for 3 h at 48˚C, and washed and dried as before. Fluorescent images of hybridized arrays were acquired using ScanArray and extracted using QuantArray (Perkin Elmer). The same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in each study (Cy3 PMT 78-80, Cy5 PMT 72-76).
GRASP microarray studies were conducted using liver samples. To minimize technical variability within a study, all targets were synthesized in one round, and all hybridizations were conducted simultaneously on slides from a single batch (EB017). Total RNA, prepared from flash-frozen adult liver tissues using TRIzol reagent and methods (Invitrogen), was quantified and quality-checked by spectrophotometer and agarose gel. The microarray experiments used pooled RNA templates: pooled C contained equal quantities from 10 individual C fish, pooled R contained equal quantities from 10 individual R fish, and pooled T contained equal quantities from 10 individual T fish. Microarray experimental design involved 3 comparisons (C vs. R, C vs. T, R vs. T), each run in duplicate (1 chip and a dye flip). Microarray hybridizations were performed using the Genisphere Array50™ kit and instructions. Briefly, 20 ug total RNA (pooled C, pooled R, or pooled T) were reverse transcribed using oligo d(T) primers with 3’ or 5’ unique sequence overhangs for the Cy3 or Cy5 labeling reactions, respectively. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95˚C MilliQ H2O, and drying by centrifugation (5 min 2000 rpm in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 48˚C water bath. The coho salmon liver cDNAs were hybridized to the salmonid cDNA microarray in a formamide-based buffer (25% formamide, 4X SSC, 0.5% SDS, 2X Denhardt's solution) 16 h at 48˚C. Coverslips were floated off in 48˚C (2X SSC, 0.1% SDS), and arrays were washed 1 X 10 min in 2X SSC, 0.1% SDS at 48˚C, 2 X 5 min in 2X SSC, 0.1% SDS at room temperature (RT), 2 X 5 min in 1X SSC at RT, and 2 X 5 min in 0.1X SSC at RT, and dried by centrifugation as before. The Cy3 and Cy5 3-dimensional fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized to the bound cDNA on the microarray in a formamide-based buffer (25% formamide, 4X SSC, 0.5% SDS, 2X Denhardt's solution) for 3 h at 48˚C, and washed and dried as before. Fluorescent images of hybridized arrays were acquired using QuantArray (Perkin Elmer). The same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in each study (Cy3 PMT 78-80, Cy5 PMT 72-76).
