Lean and fat chicken lines
The NEAU broiler lines divergently selected for abdominal fat content (NEAUHLF) have been selected since 1996 using percentage abdominal fat (%AFW) and plasma very low-density lipoprotein (VLDL) concentration as selection criteria. The G0 generation of the two lines came from the same grandsire line originating from the Arbor Acres breed, which was then divided into two lines according plasma VLDL concentration at 7 weeks. From G1 to G8, birds of each line were raised in two hatches. They were kept in similar environmental conditions and had access to feed and water ad libitum, and were fed with a commercial corn-soybean-based diet that met all NRC requirements (National Research Council, 1994). From hatching to 3 weeks the birds received a starter feed (3,100 kcal ME/kg and 210 g/kg CP), and from 4 weeks to slaughter they were fed a grower diet (3,000 kcal ME/kg and 190 g/kg CP). Plasma VLDL concentrations were measured for all birds at 7 weeks. Abdominal fat weight (AFW) of the male birds in the first hatch was measured and adjusted (%AFW) for body weight (BW) after slaughtering at 7 weeks. Birds with plasma VLDL concentration and AFP lower (lean line) or higher (fat line) than the population average value were selected as candidates for breeding, considering the body weights of male birds and egg production of female birds.
Selection was continued for 8 generations, with 15 sires and 4 hens per sire in the fat line and 10 sires and 4 hens per sire in the lean line for the G0 to G5 generation, then 25 sires and 4 dams per sire in each line for generations G6 through G8. The phenotypic correlation coefficient between body weight and AFW is 0.3789 (P
Sample preparation
Birds were slaughtered at 7 weeks and abdominal fat was isolated, immediately frozen in liquid nitrogen and stored at -80°C. The ten birds used in the present study were chosen by the percentage of abdominal fat (AFP): five had the highest AFP and the other five had the lowest (Table 1). Total RNA was extracted from 300 to 800 mg bulk abdominal adipose tissue using an RNeasy Lipid Tissue Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's recommendations, and quantified by spectrophotometry. mRNA was isolated using an Oligotex mRNA Mini Kit (QIAGEN, Valencia, CA). cDNA was prepared by oligo-dT-primed reverse transcription using Superscript II (Life Technologies, Inc.). Labeled cRNA probes were prepared using an IVT Labeling Kit (Affymetrix, Inc.) according to the manufacturer's protocol.
Microarrays, hybridization and scanning
The GeneChip Chicken Genome Array used in present study was created by Affymetrix Inc. at the end of 2004, with comprehensive coverage of over 38,000 probe sets representing 32,773 transcripts corresponding to over 28,000 chicken genes. The Chicken Genome Array also contains 689 probe sets for detecting 684 transcripts from 17 avian viruses. Sequence information for this array was selected from the following public data sources: GenBank, UniGene (Build 18; May, 2004) and Ensembl (version 1, released on May, 2004).
Twenty micrograms of cRNA were fragmented at 94°C for 35 min in a 5 × fragmentation buffer containing 200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc. Prior to hybridization, the fragmented cRNA was heated at 95°C for 5 min in 1 × MES hybridization buffer (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween20) and 0.1 mg/ml herring sperm DNA, then at 45°C for 5 min, before loading on to the Affymetrix probe array cartridge (Affymetrix, Inc.). After prehybridization in 300 μl 1 × Hybridization Buffer at 45°C for 10 min, the Chicken Genome Arrays were incubated for 16 h at 45°C at constant rotation (60 rpm) using the manufacturer's hybridization buffer. Following hybridization, the arrays were washed with 6 × SSPE-T (0.9 M NaCl/60 mM NaH2PO4/6 mM EDTA/0.01% Tween20) at 25°C on a fluidics station (Affymetrix) for 10 × 2 cycles, then washed with 0.1 M MES/0.1 M NaCl/0.01% Tween20 at 50°C for 4 × 15 cycles. The arrays were stained with a streptavidin-phycoerythrin conjugate (Molecular Probes, Eugene, OR), followed by 10 × 4 wash cycles. To enhance the signals, the arrays were further stained with anti-streptavidin antibody for 10 min followed by a 10-minute staining with the streptavidin-phycoerythrin conjugate. After 15 × 4 additional wash cycles, the arrays were scanned at 560 nm using a confocal scanner (Affymetrix Gene array Scanner3000).
Microarray data normalization
Raw data sets were normalized to total fluorescence, which represents the total amount of cRNA hybridized to a microarray, using the software Affymetrix Microarray Suite 4.0. Data sets were excluded if the absolute call (Abs Call) was A (absent) or M (Marginal) according to the detection p-value in all arrays. Only expressed transcripts (the Abs Call was P, present) were used in further analysis.
The raw data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus and have been assigned the following GEO accession numbers: GSM197816, GSM197817, GSM197818, GSM197819, GSM197820, GSM197821, GSM197822, GSM197823, GSE197824 and GSE197825
Analysis of gene expression profile in 7-week broilers
The gene expression profile was investigated by three methods. First, according to the data normalization and Affymetrix Microarray Suite 4.0 software results, a gene was considered "not detected" by the scanner if its absolute call was A; we call these genes "not expressed" in this paper. The distribution of the expressed transcript (the Abs Call was P) was characterized by JMP4.0 according to its expression level, and genes with high and low expression levels were defined on the basis of this result.
Hierarchical clustering analysis of the variation within each line
The fat and lean chicken lines have been selected for eight generations, so it is expected that the differences among individuals within each line are slight. Hierarchical clustering analysis of the genes expressed in five birds from each line was conducted to measure the consistency within each line. The value of R (coefficient correlation) was calculated and used to evaluate inter-line variation. Any individual that deviated too much from the others was excluded from the subsequent screening for differentially expressed genes.
Identification of differentially expressed genes
Differentially expressed genes were identified from normalized data using the Significance Analysis of Microarrays (SAM) algorithm [8] implemented in a TIGR MultiExperiment Viewer. According to the SAM algorithm, genes are identified as differentially expressed on the basis of expression differences among the sample groups and the consistency of these differences; a score is assigned to each gene on the basis of a change in its expression relative to the standard deviation of repeated measurements for that gene. A gene is deemed 'significant' if its score surpasses a certain threshold. SAM calculates a false discovery rate (FDR), which is the median percentage of genes that are likely to be identified as significantly changed by chance. The threshold can be adjusted to identify different sets of putatively significant genes and the FDR is changed accordingly. The number of significantly changed genes in each experiment depends on a threshold with an acceptable FDR, selected by the investigator [8,30,31]. Clustering was achieved using uncentered Pearson correlations and average linkage clustering, and was displayed in TreeView. In the present study, a ?-value of 0.8 was chosen, giving a reasonable cutoff of 3.13 and 4.73 in d-scores.
Real-time quantitative RT-PCR
Oligonucleotide primers were designed to amplify a fragment containing sequences from two adjacent exons in order to avoid contamination with genomic DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered to be a stably expressed housekeeping gene and was used as an internal reference gene. The primer pairs used to analyze gene expression and the size of product are shown in Table 6.
cDNA was prepared using an RNA PCR Kit (AMV) Ver3.0 (Takara) starting with 500 ng of total RNA from the following samples: (1) fat line: 7-3-3, 7-3-8, 7-3-15, 7-3-16, 7-3-24; (2) lean line: 7-1-1, 7-1-2, 7-1-4, 7-1-11, 7-1-17; (3) four RNA pools (two from each line), each containing three samples that were not used for the array experiments. First-strand cDNA synthesis was performed at 42°C for 30 min. The 10 μl reaction mixture also contained 1 × AMV reaction buffer, 1 mM each dNTP, 0.125 pmol oligo-dT-adaptor primer, 5 mM MgCl2, 10 U RNase inhibitor, and 2.5 U avian myeloblastosis virus (AMV) reverse transcriptase XL. After incubation, the mixture was heated at 99°C for 5 min to extinguish reverse transcriptase activity. Relative quantification of the expression of selected genes was performed using SYBR® Premix Ex Taq™ (Takara). The reaction mixtures were incubated in an ABI Prism 7300 Sequence Detection System (Applied Biosystems) programmed to conduct one cycle at 95°C for 10 s and 40 cycles at 95°C for 5 s and 60°C for 31 s. The dissociation curves were analyzed using Dissociation Curve 1.0 software (ABI) for each PCR reaction to detect and eliminate possible primer-dimer artifacts. Results (fold changes) were expressed as 2ΔΔCt with ΔΔCt = (Ctij - CtGAPDHj) - (Cti1 - CtGAPDH1), where Ctij and CtGAPDHj are the Ct for gene i and for GAPDH in a pool or a sample (named j), and Cti1 and CtGAPDH1 are the Ct in pool 1 or sample 1, expressed as the standard [9].
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