Real-time PCR assay
Total RNA from either snap-frozen muscles or cultured cells was isolated
using an RNeasy Micro Kit (Qiagen, Hilden, Germany). The mRNA was
reverse-transcribed into cDNA with PrimeScript RT Master Mix (TaKaRa,
Otsu, Japan) and subjected to quantitative real-time PCR with SYBR Green
PCR Master Mix (Life Technologies,New York,NY). The relative
expression of the target genes was normalized to that of GAPDH and
calculated with the ΔΔCt method. The following primers were used:
MuRF-1: forward: 5’-ACCTGCTGGTGGAAAACATC-3’, reverse:
5’-AGGAGCAAGTAGGCACCTCA-3’; Atrogin-1: forward:
5’-ATTCTACACTGGCAGCAGCA-3’, reverse: 5’-TCAGCCTCTGCATGATGTTC-3’; GAPDH:
forward: 5’-ACCCTTAAGAGGGATGCTGC-3’, reverse:
5’-CGGGACGAGGAAACACTCTC-3’.