Atlantic salmon of Connecticut River stock obtained as parr from the White River National Fish Hatchery in fall 2005 were reared at the Conte Anadromous Fish Research Center in Turners Falls, MA. Fish were held in 1.5 m diameter fiberglass tanks receiving flow-through (4 L min-1) Connecticut River water, maintained under natural photoperiod conditions. Fish were reared under ambient river temperatures through the fall and then at 10 (+ 1)C from February through the remainder of the study. Fish were fed to satiation twice daily with commercial feed. In late January, fish were divided into two groups and reared in separate tanks; fish smaller than 11 cm (parr) and greater than 12 cm (smolts). On April 13, 2006, fish were euthanized in 200 mg/L MS-222 neutralized to pH 7.0 and gill, liver, pituitary, hypothalamus and olfactory rosette tissues were collected in individual tubes and frozen at -80C for microarray analyses.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Qiagen RNeasy kits according to manufacturer instructions; all samples were treated with RNase-free DNase.
Label
Alexa555
Label protocol
Pituitary and hypothalamus RNA were amplified and then labeled using the Ambion Amino Allyl MessageAmpII aRNA amplification kit according to manufacturer’s instructions. 100ng or 200ngRNA was reverse transcribed into complementary DNA (cDNA) using an oligo(dT) primer containing a T7 promoter, then copied to create double-stranded DNA. In a single round of amplification, this double-stranded DNA was used as a template to synthesize amino-allyl modified RNA (aRNA) with a T7 RNA polymerase in an 8 hour in vitro transcription reaction. 5µg of amino-allyl modified, amplified RNA was labeled with Alexa Fluor 555 or 647 dyes. Liver, gill, and olfactory rosette RNA samples were not amplified prior to labeling. For these samples, 10ug total RNA was reverse transcribed and labeled using the Invitrogen Superscript Plus Indirect cDNA Labeling System and Invitrogen Alexa Fluor 555 or 647 dyes according to manufacturer’s instructions, with the following minor modifications: Qiagen Qiaquick PCR Purification spin columns were used to clean the cDNA following first strand synthesis and following labeling with Alexa Fluor dyes.
Atlantic salmon of Connecticut River stock obtained as parr from the White River National Fish Hatchery in fall 2005 were reared at the Conte Anadromous Fish Research Center in Turners Falls, MA. Fish were held in 1.5 m diameter fiberglass tanks receiving flow-through (4 L min-1) Connecticut River water, maintained under natural photoperiod conditions. Fish were reared under ambient river temperatures through the fall and then at 10 (+ 1)C from February through the remainder of the study. Fish were fed to satiation twice daily with commercial feed. In late January, fish were divided into two groups and reared in separate tanks; fish smaller than 11 cm (parr) and greater than 12 cm (smolts). On April 13, 2006, fish were euthanized in 200 mg/L MS-222 neutralized to pH 7.0 and gill, liver, pituitary, hypothalamus and olfactory rosette tissues were collected in individual tubes and frozen at -80C for microarray analyses.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Qiagen RNeasy kits according to manufacturer instructions; all samples were treated with RNase-free DNase.
Label
Alexa647
Label protocol
Pituitary and hypothalamus RNA were amplified and then labeled using the Ambion Amino Allyl MessageAmpII aRNA amplification kit according to manufacturer’s instructions. 100ng or 200ngRNA was reverse transcribed into complementary DNA (cDNA) using an oligo(dT) primer containing a T7 promoter, then copied to create double-stranded DNA. In a single round of amplification, this double-stranded DNA was used as a template to synthesize amino-allyl modified RNA (aRNA) with a T7 RNA polymerase in an 8 hour in vitro transcription reaction. 5µg of amino-allyl modified, amplified RNA was labeled with Alexa Fluor 555 or 647 dyes. Liver, gill, and olfactory rosette RNA samples were not amplified prior to labeling. For these samples, 10ug total RNA was reverse transcribed and labeled using the Invitrogen Superscript Plus Indirect cDNA Labeling System and Invitrogen Alexa Fluor 555 or 647 dyes according to manufacturer’s instructions, with the following minor modifications: Qiagen Qiaquick PCR Purification spin columns were used to clean the cDNA following first strand synthesis and following labeling with Alexa Fluor dyes.
Hybridization protocol
Microarray slides from the same print batch were used for each tissue comparison. The print batch for the microarrays used in the gill, olfactory rosettes, hypothalamus, and pituitary comparisons had an unusable subarray (column 1, row 8) due to printing errors. Prior to hybridization, microarray slides were washed ten minutes in 0.1% SDS, and then rinsed five minutes in ultrapure water. Slides were dried by centrifugation and pre-warmed at 50C for 30 minutes. For each slide, 15 pmol of the Alexa 555 labeled aRNA or cDNA and 15 pmol of the Alexa 647 labeled aRNA or cDNA were combined and mixed with Ambion SlideHyb no. 2 to a volume of 60 µl for hybridization. The combined samples and hybridization buffer were heated at 80C for ten minutes and pipetted under a Erie Scientific LifterSlip coverslip on top of the microarray slide. The slides were incubated at 50C, shaking slowly, for 16-18 hours in a Boekel Scientific Shake ‘N’ Bake hybridization oven. After hybridization, slides were washed on an orbital shaker for ten minutes in heated 2x SSC + 0.5% SDS; coverslips floated off the slides during this wash. Slides were then washed for 5 minutes in heated 2x SSC + 0.5% SDS, five minutes in 0.5x SSC, and five minutes in 0.05x SSC. Slides were dried by centrifugation and immediately scanned.
Scan protocol
Slides were scanned at 10 µm resolution in a Perkin Elmer ScanArray Gx scanner. Laser power and PMT voltage were adjusted using the Line Scan Protocol such that the signal from the Alexa Fluor 555 dye was approximately equal to the signal from the Alexa Fluor 647 dye for each slide and the signal was saturated for approximately 0.3-0.5% of the features on the slide.
Description
biological replicate 6 of 6
Data processing
Spot-finding was done with ScanArray Express Microarray Analysis System software version 4.0 using the adaptive circle method. Median spot intensities and median background intensities were imported into the TM4 Microarray Software using the Express Converter version 2.1 utility; median background intensities were subtracted from median spot intensities. Converted intensity files were imported into TM4 Midas version 2.20 with RMA and spots where the background-corrected intensity was less than zero for either channel (dye) were removed. The log2 intensity ratios for each array were normalized using a local Lowess normalization (by subarray) and standard deviation regularization for the blocks.
