Lee et al. BMC Genomics 2010, 11:623 http://www.biomedcentral.com/1471-2164/11/623 Page 9 of 11 (10 μl) (Qiagen., GmbH, Germany), and 10 pM for- ward and reverse primers. The real-time PCR reactions started at 95°C for 15 min for pre-denaturation and the condition was set at 95°C for 10 s, 56°C for 20 s and 72°C for 30 s. The PCR performed 40 cycles. The PCR was conducted in ABI 7500 realtime PCR system (Applied Biosystems, USA). Primer sets used in the real-time PCR are listed in Table 5. The relative gene expression value was calculated by the ΔCt method [36]. The ΔCt value of the target gene was normalized against the G3PDH Ct value. The fold change was determined as 2-ΔCt. For selected genes in the putative ADAMTS4 pathway, a regression analysis of gene expression value (2-ΔCt) on intramuscular fat and protein content was performed using a simple linear regression in R [28]. Table 5 Primer sequences used in qRT-PCR Probe ID Gene Name Bt.5323.1. S1_at Bt.15675.1. S1_at Bt.21021.1. S1_at Bt.2933.1. S1_at Bt.9767.1. S1_a_at Bt.621.1. S1_at Bt.23903.1. A1_at Bt.22362.1. S1_at Bt.16752.1. A1_at Bt.1020.1. S1_at Bt.19107.2. A1_at Bt.28011.1. S1_at Bt.22718.1. A1_at Bt.19107.1. S1_at Bt.25102.1. S1_a_at Bt.22038.1. S1_a_at Bt.21268.1. S2_at Bt.13342.1. S1_at Bt.344.1. S1_at Bt.21827.2. S1_at Bt.21794.1. S1_at G3PDH IL17A TNFa TGFb1 SH3 domain YSC-like 1 (SH3YL1) ADAM metallopeptidase with thrombospondin motif 4 (ADAMTS4) TBC1 domain family, member 7 (TBC1D7) Hypothetical protein LOC788205 Squalene epoxidase (SQLE) Cytochrome P450, family 51, subfamily A (CYP51A) Unknown SH3-domain kinase binding protein 1 (SH3KBP1) ATP binding protein (TXNDC9) CDC-like kinase 1 (CDClk1) Transcribed locus Unknown Proteasome (prosome, macropain) activator subunit 4 Transcribed locus Hypothetical LOC509649 Arginyl-tRNAsynthetase (RARs) Thimetoligopeptidase 1 (THOP1) Hypothetical protein LOC777601 Glyceride 3 phosphatate dehydrogenase Interleukin 17A Tumor necrosis factor Transforming growth factor beta 1 NM_181667 AGTTCGACAAGTGCATGGTGTGTG TGGTGACCACGTTGTTGTATCCGT NCBI accession no. CB535095 CB451394 CK972377 CK949309 BE664559 BP102962 CK774919 CB536841 CK848317 BE723026 CK940528 CK945034 BE723026 CK772143 CK947459 D76416.1 CB444022 CA034934 AY779626 CB432107 CK848164 CK772652 Forward primer (5’->3’) Reverse primer (5->3’) ACCAACCCATAGAAGTGACAGCAC CGAAGCTTTCCTTCCCACCAATCA CTTCGTGAACCAGCTGAACAGCAA CGGCAAAGCACTTCTTGAACCACA GCCAAAGCAGCTGTCGGTAATGAA TCCATCACACCGCGAAGACTCTAA AGTAATCATCGTGGGATCTGGCGT ACCTGGGCATCAATACCTTCCACT GTATGACCTCAACAACCCTGCCAA TGACCACGACGATGATGAAGACCA ACACAGGCCGTGCAAACTAAACAC TCTTGATTTGCTGCTGGGACCTCT TGAGGGATGCACAGATGAGTGTGA TTGAAGGCTGGAGGGCACATCTTT TTCATCTGCTGATGGCCACACTC AGAGAATCCAGACTCTCCTCAGA TTTAGGATGGTCCCAGCCATGTGA GCCCAAGAATCCTTTCCATCATTGCC CCATGAGAACTGACTCGGGAGTTT CAGGCTGTCTGGCAAGCAACAATA AGGAAGAACCTTCTGTCCCAGCTT TTGAGACTTCCCAGGTCAAAGGCA GCTGAGGTGTGGGTTTGTTTGAGT AAACTGGTCACAGGCAAACACG GACCCAAGAGTTGCTTAAGAGAGC ACCCTCAGTCCACAGATGATCAAG TTCCTCCCACTGGTGAGCATCTTT TGTGTTGCTCAGTGTTCTCCTCCA TTGAAGGCTGCTCAGACCTCTGTT TAAGCCGCTGTGTTTCCTCTGTCA AAGGTCTCCATCTGGAGGTGTTTG AACTCCCAGGAAAGGGCTGCATT CATGAGACACAGGCGAAACACTGA TCTTTGGGAGAAAGGGAAACTGGG GGGTCATCATCTCTGCACCT TCATCATCCCACAGAGTCCA GGCCATGGTATTGACATCCT ACTACATCTCGGCGCTCAGT GGTCATAAGTCCCTCCACGA GGAGAGTCCAAGGTGAGGTG GGATCTTCTCCACCACATCG GAAGGTGCAGGTGAAGTGGT Ribosomal protein S6 kinase, 70kDa (TUBD1) CK977623 CGTGACTGTAGATGGTGAAAGGGT TGCACACTCAGACTGAAGACAC Src-associated protein SAW (UTP15) BM030756 CTCATAGCCATCAATAGTTCAGTGC TCAAGTAGCAAATACTACAGTTTGTC Major histocompatibility complex, class II TCACACCAGCACCCTCTGATCTTT TAAGCACGGCTTTCGGCAGTAGAA