Lee et al. BMC Genomics 2010, 11:623 http://www.biomedcentral.com/1471-2164/11/623 Methods Animals and sampling The two divergent groups were formed by selecting the 5 highest and the 5 lowest marbling score carcasses from a population of 90 steers of pure bred Hanwoo cattle aged between 28 and 30 months old raised under similar conditions. Intramuscular fat (IMF %) of the muscle samples were measured using chemical fat extraction procedures and crude protein was measured on extracted samples (1 to 1.5 g) using the Macro- Kjeldahl method [27]. Table 1 presents summary statis- tics for all muscle samples used in this study. Total RNA isolation and Microarray hybridization From each animal, after removal of intramuscular fat from the samples, total RNA samples were prepared from 1 g of frozen m. longissimus tissue using Trizol reagent (Invitrogen Inc., USA) according to the manu- facturer’s instructions. Messenger RNA was isolated from 500 μg of total RNA using Qiagen Oligotex resin (Qiagen Inc, USA). The quantity and quality of RNA samples were measured using absorbance at 260 nm and 280 nm in a capillary type spectrophotometer (Agi- lent Inc, USA) and confirmed in 1.2% formaldehyde- contained gel electrophoresis. Target preparation and high-density array hybridization Double stranded cDNA was synthesized from 3 μg mRNA using a Genechip Expression 3’-Amplification One Cycle Synthesis kit (Affymetrix Inc. USA). The cDNA was purified using a Genechip Sample Cleanup Module (Affymetrix Inc). Biotin-labeled cRNA was synthesized in vitro using the Gene chip Expression 3’- Amplification reagents in the IVT labeling kit (Affyme- trix Inc.). Biotin-labeled antisense cRNA was purified using the Genechip Sample Cleanup Module (Affymetrix Inc.) and the cDNA was fragmented in the 5 × Fragmen- tation buffer provided with the Genechip Sample Cleanup Module (Affymetrix Inc). Hybridization cocktail (200 μl) containing 15 μg fragmented cRNA was injected into the Genechip Bovine Genome Array (Affymetrix Inc). The array was placed in a 45 degrees hybridization oven at 60 rpm for 16 hours. After hybridization, the arrays were washed and stained in a fluidic station with the appropriate signal amplification protocol using bioti- nylated anti-streptavidin antibody (Vector Laboratories Inc., USA) and phycoerythrin-conjugated streptavidin (Invitrogen Inc., Carlsbad, CA, USA). The array was scanned with a GeneChipScanner 3000 (Affymetrix Inc.). Microarray Data processing All quality control measures, preprocessing and analyses were performed using the statistical computing language Page 8 of 11 R [28]. The quality of the arrays was assessed through standard quality control measures for Affymetrix arrays: pseudoimages of the arrays, MA scatter plots of the arrays versus a pseudomedian reference chip and other summary statistics including histograms and box plots of raw log intensities, box plots of relative log expressions, box plots of normalized unscaled standard errors and RNA degradation plots [29]. Transcription intensities in log2 scale were estimated from the probe-level data by using three summarization methods: MAS5.0 [30], RMA [30] and GCRMA [31]. In MAS5.0, each probe was adjusted using a weighted average. All arrays were scaled to the same mean value for normalization (200) and were summarized by a log2 scale average using 1-step Tukey biweight. For RMA, the background was corrected by convolution. The data were quantile normalized and summarized by median polish. GCRMA background cor- rection used an affinity measure model based on probe sequences and mismatch intensities. Presence calls [32] for the probes were also calculated (τ = 0.015, a1 = 0.04 and a2 = 0.06). The data were filtered to remove control probes (n = 133) and probes detected as marginal or absent in all arrays using MAS5 presence calls. Microarray data analysis Prior to testing for differential expression, the data were filtered to remove Affymetrix control probes (n = 133) and all noninformative probes detected as marginal or absent in all arrays (n = 11,383), thus remaining 12,745 probes to be tested. Differential transcription was tested for each summarization method using LIMMA [33,34]. Only differentially expressed (DE) probes detected in two out of the three summarization methods (P < 0.01) and flagged as present in at least 50% of the samples were con- sidered to be significant. This approach ensures maximum specificity to detect differential expression and minimizes the effect of different summarization methods which are the main source of variability in the analysis of Affymetrix arrays. No false discovery rate correction method is war- ranted due to the stringency of the filtering criteria [35]. QRT-PCR for the 21 DEGs and selected genes from the ADAMTS4 pathway For technical validation of the microarray results, the 21 differentially expressed genes were validated by quantitative realtime RT-PCR (qRT-PCR). cDNA synthesis was performed using 2 μg of total RNA. The complimentary strand was primed with a random pri- mer (Promega.co) and cDNA synthesis was performed using a Superscript III kit (Invitrogen) according to the manufacturer’s instructions. Each quantitative PCR was carried out in a final volume of 20 containing 1 μl cDNA (500 ng/μl), 2 × SYBR Green I Master Mix