Quantitative Analysis of Muscle Gene Expression

Total RNA was extracted from the breast muscle using RNAiso (Takara Bio, Dalian, Liaoning, China). The first-strand cDNA was synthesized using the SuperScript® III Reverse Transcriptase with random primers and an RNase inhibitor (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions. Gene-specific primers for IGF-1, MSTN, MyoD1, myogenin, MRF4, Myf5, MEF2A, MEF2B, MEF2C, and MEF2D were designed using Primer Premier 6.0 (Premier, Ontario, Canada) (Table 1). PCR was performed on the ABI 7500 Real-Time PCR Detection System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq II Kit (Takara Bio) and 40 cycles of 95 °C for 15 s and 60 °C for 30 s. All measurements were performed in triplicate. The fold difference was calculated using the ∆∆Ct method [10 (link), 11 (link)] using the geometric means of 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA for data normalization.Table 1

Primers Used for Real-Time PCR

Gene	GenBank Accession Number	Primer Sequence (5' to 3')	Product Size, bp
IGF-1	NM_001004384	GAGCTGGTTGATGCTCTTCAGTT	148
		CCAGCCTCCTCAGGTCACAACT
MSTN	NM_001001461	CGCTACCCGCTGACAGTGGAT	132
		CAGGTGAGTGTGCGGGTATTTCT
MyoD1	NM_204214	CCGACGGCATGATGGAGTACA	131
		GTCGAGGCTGGAAACAACAGAA
Myogenin	D90157	GGAGGCTGAAGAAGGTGAACGA	127
		CTCTGCAGGCGCTCGATGTACT
MRF4	D10599	CAGGCTGGATCAGCAGGACAA	106
		GCCGCAGGTGCTCAGGAAGT
Myf5	NM_001030363	CAGAGACTCCCCAAAGTGGAGAT	106
		GTCCCGGCAGGTGATAGTAGTTC
MEF2A	NM_204864	CGGAGGACAGATTCAGCAAACTA	109
		GACACTGGAACCGTAACCGACAT
MEF2B	XM_430389	CACGCCATCAGCATCAAGTCA	156
		GGGGTAGCCCTTGGAGTAGTCAT
MEF2C	XM_001231661	GCCGTCTGCCCTCAGTCAACT	137
		GGGTGGTGGTACGGTCTCTAGGA
MEF2D	NM_001031600	GTGTCTCCCAAGCGACTCACTCT	109
		GTGTTGTATGCGGTCGGCAT
GAPDH	NM_204305	GCCACACAGAAGACGGTGGAT	86
		GTGGACGCTGGGATGATGTTCT
18S	AF173612	CCGGACACGGACAGGATTGACA	94
		CAGACAAATCGCTCCACCAACTAAG