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Detection of Dystrophin Dp71 in Human Skeletal Muscle Using an Automated Capillary Western Assay System
Tatsuya Kawaguchi1, Emma Tabe Eko Niba2,3, Abdul Qawee Mahyoob Rani4
1Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd., Edogawa, Tokyo 134-8634, Japan. kawaguchi.tatsuya.sg@rdn.daiichisankyo.co.jp.
Background:
Dystrophin Dp71 is one of the isoforms produced by the DMD gene which is mutated in patients with Duchenne muscular dystrophy (DMD). Although Dp71 is expressed ubiquitously, it has not been detected in normal skeletal muscle. This study was performed to assess the expression of Dp71 in human skeletal muscle.
Methods:
Human skeletal muscle RNA and tissues were obtained commercially. Mouse skeletal muscle was obtained from normal and DMDmdx mice. Dp71 mRNA and protein were determined by reverse-transcription PCR and an automated capillary Western assay system, the Simple Western, respectively. Dp71 was over-expressed or suppressed using a plasmid expressing Dp71 or antisense oligonucleotide, respectively.
Results:
Full-length Dp71 cDNA was PCR amplified as a single product from human skeletal muscle RNA. A ca. 70 kDa protein peak detected by the Simple Western was determined as Dp71 by over-expressing Dp71 in HEK293 cells, or suppressing Dp71 expression with antisense oligonucleotide in rhabdomyosarcoma cells. The Simple Western assay detected Dp71 in the skeletal muscles of both normal and DMD mice. In human skeletal muscle, Dp71 was also detected. The ratio of Dp71 to vinculin of human skeletal muscle samples varied widely, indicating various levels of Dp71 expression.
Conclusions:
Dp71 protein was detected in human skeletal muscle using a highly sensitive capillary Western blotting system.
Insights
Dystrophin Dp71 protein was detected in human skeletal muscle, a finding previously unobserved. This study utilized sensitive capillary Western blotting to confirm Dp71 expression in skeletal muscle tissue.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Dystrophin Dp71 is an isoform of the DMD gene, implicated in Duchenne muscular dystrophy (DMD).
- Dp71 is ubiquitously expressed but not typically found in normal skeletal muscle.
- This study investigates Dp71 expression in human skeletal muscle.
Purpose of the Study:
- To assess the presence and expression levels of Dp71 protein in human skeletal muscle.
- To validate Dp71 detection methods in skeletal muscle tissue.
Main Methods:
- Reverse-transcription PCR (RT-PCR) for Dp71 mRNA detection.
- Automated capillary Western assay (Simple Western) for Dp71 protein quantification.
- Over-expression and antisense oligonucleotide suppression in cell lines for protein validation.
- Analysis of skeletal muscle from normal and DMD mice.
Main Results:
- Full-length Dp71 cDNA was successfully amplified from human skeletal muscle RNA.
- The Simple Western assay confirmed a ca. 70 kDa protein as Dp71.
- Dp71 protein was detected in skeletal muscles of both normal and DMD mice, and in human skeletal muscle.
- Significant variability in Dp71 to vinculin ratios was observed in human samples.
Conclusions:
- Dp71 protein is detectable in human skeletal muscle.
- A sensitive capillary Western blotting system effectively detected Dp71 in skeletal muscle.
- The findings suggest a potential role or presence of Dp71 in skeletal muscle not previously characterized.
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