Reiko Nakao1, Saori Yamamoto, Yuki Yasumoto, Koji Kadota, Katsutaka Oishi. 1. Biological Clock Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
Abstract
INTRODUCTION: Immobilization induced by experimental denervation leads to rapid and progressive alterations in structural and biochemical properties of skeletal muscle. Real-time reverse transcription-polymerase chain reaction (RT-PCR) is a popular method of elucidating the molecular mechanisms involved in muscle atrophy. Identification of suitable reference genes that are not affected by experimental conditions is a critical step in accurate normalization of real-time RT-PCR. METHODS: We investigated the impact of denervation-induced muscle atrophy for 2 weeks on the expression of common housekeeping genes. RESULTS: Denervation differentially affected the expression levels of these genes. RefFinder software identified TATA box binding protein (Tbp) as the most stable gene and showed that the stability of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and hypoxanthine guanine phosphoribosyl transferase (Hprt) genes was low, even though they are widely used for normalization. CONCLUSIONS: The appropriate reference gene for normalization of genes of interest in denervated muscle is Tbp.
INTRODUCTION: Immobilization induced by experimental denervation leads to rapid and progressive alterations in structural and biochemical properties of skeletal muscle. Real-time reverse transcription-polymerase chain reaction (RT-PCR) is a popular method of elucidating the molecular mechanisms involved in muscle atrophy. Identification of suitable reference genes that are not affected by experimental conditions is a critical step in accurate normalization of real-time RT-PCR. METHODS: We investigated the impact of denervation-induced muscle atrophy for 2 weeks on the expression of common housekeeping genes. RESULTS: Denervation differentially affected the expression levels of these genes. RefFinder software identified TATA box binding protein (Tbp) as the most stable gene and showed that the stability of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and hypoxanthine guanine phosphoribosyl transferase (Hprt) genes was low, even though they are widely used for normalization. CONCLUSIONS: The appropriate reference gene for normalization of genes of interest in denervated muscle is Tbp.
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