Total RNA was isolated from RNAlater (Ambion), stabilized oyster tissues using Qiagen's RNeasy Mini kits with an on- column DNase (Qiagen) treatment. Extracted RNA was quantified by absorbance at 260nm using a NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies).
Label
Cy3
Label protocol
One microgram of total RNA was used to produce Cy3-labeled aminoallyl RNA (Cy3-aRNA) probe using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion), according to the manufacturer's instructions.
Hybridization protocol
Ten micrograms of the subsequently produced Cy3-aRNA was diluted (1:3) in hybridization buffer (50% formamide, 2.4% SDS, 4X SSPE, 2.5X Denhardt's solution, and 2µl of blocking solution (1µg Cot-1 DNA and 1µg poly (dA)). The probe was then boiled for 1 min and incubated in the dark for 1 h at 50°C prior to hybridization. The slides were prewashed with 0.2% SDS for 2 min, just-boiled Milli-Q water for 2 min, rinsed in 70% ethanol for 2 min, and then dried. Slides were pre-hybridized in a hybridization oven with a pre-hybridization buffer (33.3% formamide, 1.6% SDS, 2.X SSPE, 1.6X Denhardt’s solution, and 0.1µM salmon sperm DNA) in the dark for 1 h at 50°C. Slides were then hybridized with Cy-3-aRNA in the dark for 16 h at 50°C. After the hybridization, slides were rinsed in 2X SSC, 0.1% SDS and soaked in 0.2X SSC, 0.1% SDS for 15 min in the dark at room temperature, followed by a rinse in 0.2X SSC, soaking in 0.2X SSC for 15 min, 0.1X SSC for 15 min and finally Milli-Q water for 5 min in the dark in order to remove carryover SDS.
Scan protocol
The micorarray were scanned using ScanArray Express and SpotArray software at 70 V photomultiplier tube (PMT) gain and the raw data was quantified with QuantArray software (Perkin Elmer).
Description
050-G
Data processing
The data were background corrected using the normexp method and a print-order loess normalization was carried out. Next, genes which the 4 replicates did not show intensity higher than three times the average intensity of the non-oyster genes present in the slides in at least 10% of the slides in the experiment were disregarded. Last, the remaining data were variance stabilization normalized (vsn).
