tissue: pectoralis major muscle line: F (selected from RBC2 line for increased 16wk body weight) age: 18-day embryo
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using TRIReagent (Molecular Research Center, Cincinnati, OH) following manufacturer's instructions. RNA integrity and concentration was determined using an Agilent 2100 Bioanlyzer (Santa Clara, CA).
Label
Cy5
Label protocol
Total RNA for use in microarrays was amplified using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion, Inc.) per manufacturer instructions. Briefly, 2 μg total RNA was incubated with T7 Oligo(dT) primer for 10 min at 70°C then reverse transcribed into cDNA at 42°C for 2 h. Second strand synthesis was performed at 16°C for 2 h. Double-stranded cDNA was then purified, and in vitro transcription was performed at 37°C for 4 h. Amplified RNA (aRNA) was purified and quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). For the dye coupling procedure, 10 μg aRNA was coupled to either Cy3 or Cy5 fluorescent dye (GE Healthcare, Piscataway, NJ) in the dark at room temperature for 1 hour until the reaction was quenched by the addition of 4M hydroxylamine. Dye-coupled aRNA was purified and quantified, then 5 μg was fragmented to 60-200 nucleotide segments using RNA Fragmentation Reagents (Ambion, Inc.) at 70°C for 15 min in preparation for microarray hybridization.
Channel 2
Source name
Turkey pectoralis major muscle, F, 1-day post-hatch chick
tissue: pectoralis major muscle line: F (selected from RBC2 line for increased 16wk body weight) age: 1-day post-hatch chick
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using TRIReagent (Molecular Research Center, Cincinnati, OH) following manufacturer's instructions. RNA integrity and concentration was determined using an Agilent 2100 Bioanlyzer (Santa Clara, CA).
Label
Cy3
Label protocol
Total RNA for use in microarrays was amplified using the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion, Inc.) per manufacturer instructions. Briefly, 2 μg total RNA was incubated with T7 Oligo(dT) primer for 10 min at 70°C then reverse transcribed into cDNA at 42°C for 2 h. Second strand synthesis was performed at 16°C for 2 h. Double-stranded cDNA was then purified, and in vitro transcription was performed at 37°C for 4 h. Amplified RNA (aRNA) was purified and quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). For the dye coupling procedure, 10 μg aRNA was coupled to either Cy3 or Cy5 fluorescent dye (GE Healthcare, Piscataway, NJ) in the dark at room temperature for 1 hour until the reaction was quenched by the addition of 4M hydroxylamine. Dye-coupled aRNA was purified and quantified, then 5 μg was fragmented to 60-200 nucleotide segments using RNA Fragmentation Reagents (Ambion, Inc.) at 70°C for 15 min in preparation for microarray hybridization.
Hybridization protocol
Fragmented, Cy3-coupled aRNA was mixed with its Cy5-coupled partner, and the mixtures were brought to 110 μL with 68°C SlideHyb 1 (Ambion, Inc.) and incubated at 68°C for 5 min. These dye-coupled mixtures were then hybridized to oligonucleotide microarrays (described previously) for 18 h in a GeneTac Hybridization Station (Genomic Solutions, Ann Arbor, MI) using the following conditions: 54°C for 18 h, followed by a medium-stringency wash at 42°C (2X SSC, 0.1% SDS), a high-stringency wash (0.2X SSC, 0.1% SDS) at 22°C, and a wash with postwash buffer (0.2X SSC) at 22°C. Arrays were then rinsed in 2X SSC and Nanopure H2O, dried by centrifugation at 500 × g for 5 min.
Scan protocol
Scanned with a GenePix 4000B (Molecular Devices, Sunnyvale, CA) scanner. Image analysis was performed using GenePix Pro 6.0, and spot intensities were exported as GPR files.
Description
F line 18d embryo versus 1d chick comparison, replicate 10 of 10.
Data processing
Fluorescence intensity data was normalized for dye intensity bias using the LOESS procedure of the normalizeWithinArray function of the Bioconductor R software LIMMA (Smyth 2004).
