tissue: anterior pituitary gland phenotype: high growth Sex: male age: posthatch week 5
Treatment protocol
Birds were divergently selected for body weight at 8 weeks of age and are maintained at SRA-INRA (France). Anterior pituitary glands were collected at 1, 3, 5, and 7 weeks of age, snap frozen in liquid nitrogen, and stored at -80oC until RNA extraction.
Growth protocol
Birds were grown using standard broiler chicken production parameters.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with the RNeasy Mini Kit according to the manufacturer's protocol (Qiagen, Valencia, CA) and 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 (aRNA) was quantified with the Ribogreen RNA Quantitation Kit (Invitrogen, Carlsbad, CA).
Label
Cy3
Label protocol
One microgram aRNA (equivalent to one microgram mRNA) of each individual sample or the reference pool was converted to cDNA using random primers with the Amino Allyl cDNA Labeling Kit (Ambion), following which Cy3- or Cy5-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). For the Cy5-labeled reference sample, a pool was made of all Cy5-labeled cDNA and split among the 32 slides in the experiment.
Channel 2
Source name
Anterior Pituitary Reference Pool (High and Low Growth lines, posthatch weeks 1, 3, 5, and 7)
tissue: anterior pituitary gland sample type: pool of High Growth and Low Growth aRNA from all 32 samples Sex: male age: posthatch weeks 1, 3, 5, and 7
Treatment protocol
Birds were divergently selected for body weight at 8 weeks of age and are maintained at SRA-INRA (France). Anterior pituitary glands were collected at 1, 3, 5, and 7 weeks of age, snap frozen in liquid nitrogen, and stored at -80oC until RNA extraction.
Growth protocol
Birds were grown using standard broiler chicken production parameters.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with the RNeasy Mini Kit according to the manufacturer's protocol (Qiagen, Valencia, CA) and 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 (aRNA) was quantified with the Ribogreen RNA Quantitation Kit (Invitrogen, Carlsbad, CA).
Label
Cy5
Label protocol
One microgram aRNA (equivalent to one microgram mRNA) of each individual sample or the reference pool was converted to cDNA using random primers with the Amino Allyl cDNA Labeling Kit (Ambion), following which Cy3- or Cy5-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). For the Cy5-labeled reference sample, a pool was made of all Cy5-labeled cDNA and split among the 32 slides in the experiment.
Hybridization protocol
The microarray was hybridized overnight at 42oC with Cy3-labeled experimental sample 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
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 25); 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. Data from each spot on each slide were expressed as log2-ratio, or log2(normalized Cy3/raw Cy5), which is given in the data table below. Log2-ratios for the spots were statistically analyzed by two-way ANOVA using a linear mixed model procedure (PROC MIXED; SAS institute Inc., Cary, NC) to identify differentially expressed genes that were significantly different (n=4; P<0.05) by line or the line-by-age interaction and where there was a >160% difference between the highest expression in one line and the lowest expression in the other.
