Divergently selected on abdominal fat weight (FL genotype) for seven generations.
Growth protocol
Standard broiler chicken growth protocol.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using a RNeasy Midi kit according to the manufacturer’s protocol (Qiagen; Valencia, CA). The RNA concentration was determined with a GneQuant II spectrophotometer (Pharmacia Biotech, Piscataway, NJ). RNA integrity was examined using a RNA 6000 Nano Assay kit and the Model 2100 Bioanalyzer (Agilent Technologies; Palo Alto, CA). Total RNA was then amplified using a modification of the Eberwine Procedure (Phillips J, Eberwine JH 1996 METHODS: A Companion to Methods in Enzymology 10: 283-288). Briefly, 500 ng was reverse-transcribed using SuperScript II (Invitrogen, Carlsbad, CA) and an oligo (dT) primer containing a T7 promoter site (5'-GGCCAGTGAATTGTAATATCGACTCACTATAGGGAGGCGGT24-3'; Affymetrix, Santa Clara, CA). Following second strand synthesis and purification, double-stranded cDNA was used as a template for in vitro transcription using the T7 MEGAscript kit (Ambion, Austin, TX) and the resulting amplified antisense RNA was quantified with the Ribogreen RNA Quantitation Kit (Invitrogen, Carlsbad, CA).
Label
Cy3
Label protocol
1 microgram amplified antisense RNA (equivalent to 1 microgram mRNA) was converted to cDNA using random primers with the Amino Allyl cDNA Labeling Kit (Ambion), and either Cy3- (test) or Cy5- (reference) monoreactive NHS esters (Amersham Biosciences, Piscataway, NJ) were coupled to the cDNA. Labeled cDNA was purified from unicorporated dye using CyScribe GFX Purification Kit (Amersham Biosciences).
Channel 2
Source name
Reference pool of pituitary total RNA from fat and lean lines, weeks 1, 3, 5, and 7
tissue: pituitary sample type: Reference pool of pituitary total RNA from fat and lean lines, weeks 1, 3, 5, and 7
Treatment protocol
Divergently selected on abdominal fat weight (FL genotype) for seven generations.
Growth protocol
Standard broiler chicken growth protocol.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using a RNeasy Midi kit according to the manufacturer’s protocol (Qiagen; Valencia, CA). The RNA concentration was determined with a GneQuant II spectrophotometer (Pharmacia Biotech, Piscataway, NJ). RNA integrity was examined using a RNA 6000 Nano Assay kit and the Model 2100 Bioanalyzer (Agilent Technologies; Palo Alto, CA). Total RNA was then amplified using a modification of the Eberwine Procedure (Phillips J, Eberwine JH 1996 METHODS: A Companion to Methods in Enzymology 10: 283-288). Briefly, 500 ng was reverse-transcribed using SuperScript II (Invitrogen, Carlsbad, CA) and an oligo (dT) primer containing a T7 promoter site (5'-GGCCAGTGAATTGTAATATCGACTCACTATAGGGAGGCGGT24-3'; Affymetrix, Santa Clara, CA). Following second strand synthesis and purification, double-stranded cDNA was used as a template for in vitro transcription using the T7 MEGAscript kit (Ambion, Austin, TX) and the resulting amplified antisense RNA was quantified with the Ribogreen RNA Quantitation Kit (Invitrogen, Carlsbad, CA).
Label
Cy5
Label protocol
1 microgram amplified antisense RNA (equivalent to 1 microgram mRNA) was converted to cDNA using random primers with the Amino Allyl cDNA Labeling Kit (Ambion), and either Cy3- (test) or Cy5- (reference) monoreactive NHS esters (Amersham Biosciences, Piscataway, NJ) were coupled to the cDNA. Labeled cDNA was purified from unicorporated dye using CyScribe GFX Purification Kit (Amersham Biosciences).
Hybridization protocol
The microarray was hybridized overnight at 42 C with Cy3-labeled test cDNA and an aliquot of the Cy5-labeled reference pool cDNA using microarray hybridization buffer (Amersham Biosciences). Slides were washed in increasing stringency using salt sodium citrate
Scan protocol
The microarray slides were scanned using a 418 confocal laser scanner (Affymetrix) at 550 nM for Cy3 and 649 nM for Cy5 to generate two TIFF images for each slide.
Data processing
The two images from each slide were processed with Spotfinder version 2.2.4 (The Institute for Genomic Research, Rockville, MD) using the following settings: Otsu thresholding algorithm (minimum spot size 3 and maximum spot size 22.5); keep flagged values; keep raw data; and quality control filter set so 50% cutoff equals background plus 1 standard deviation. Data were normalized using Microarray Data Analysis System (MIDAS) version 2.18 (The Institute for Genomic Research). In MIDAS, channel A and B flags and channel A and B background checking were used, and the signal-to-noise threshold was set at 3.0. Data from the Cy3 channel were first Lowess (LocFit) normalized by pin (block) using a smoothing parameter of 0.33 with Cy5 as the reference and then subjected to standard deviation regularization first by pin (block) then by slide with Cy5 as the reference. The data from each spot on each slide were then expressed as log2 ratio (normalized Cy3/raw Cy5), which is given in the data table below. Log2 ratios for the spots were statistically analyzed by two-way analysis of variance using Statistical Analysis Systems version 8.02 (SAS Institute, Cary, NC) to identify cDNAs that were differentially expressed between lines, among ages, and interactions between lines and ages (n = 4; p < 0.05), and a fold-change of > 1.6 was used as a filter for false positives.
