cdna of human skeletal muscle cells  (ScienCell)

Journal:

Article Title: Cloning of cDNA Encoding a Regeneration-Associated Muscle Protease Whose Expression Is Attenuated in Cell Lines Derived from Duchenne Muscular Dystrophy Patients

doi:

Figure Lengend Snippet: Identification of up- and down-regulated genes in the mdx-derived skeletal muscle cell line. A: Extent of spontaneous myogenic differentiation in the B10-sm and mdx-sm myoblastic cell lines derived from B10 and mdx mice, respectively. Cells cultured in differentiation-inducing conditions were immunostained with anti-desmin antibody. B: Northern blot analysis of various mdx up- and down-regulated genes in mdx-sm and B10-sm cell lines. The integrity and amount of loaded RNAs were assessed by probing with β-tubulin cDNA. C: RT-PCR analysis of various mdx up- and down-regulated genes in the intact skeletal muscle of B10 and mdx mice. Total RNAs were converted to cDNA by random hexamers and subjected to 20 cycles of amplification with specific primer sets for each gene as indicated. DNA bands after ethidium bromide staining are shown with GAPDH, which acts as the control that ensures equal amount of template cDNA were used.

Article Snippet: 23 By using cDNA derived from human skeletal muscle cells (Cambrex, Baltimore, MD) as the template, two polymerase chain reactions (PCRs) were independently performed with two sets of primers, namely, HD1 (5′-CTCGAGATGCTTTGGTGGGAAGAAGT-3′) and HD2(5′-TCTTTCAAGGGTATCCACAGTAATCTGCCTCTTC-3′) orHD3 (5′-ATTACTGTGGATACCCTTGAAAGACTCCAGGAAC-3′) and HD4 (5′-GCGGCCGCCTACATTGTGTCCTCTCTCAT-3′).

Techniques: Derivative Assay, Cell Culture, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Staining, Control

Journal:

Article Title: Cloning of cDNA Encoding a Regeneration-Associated Muscle Protease Whose Expression Is Attenuated in Cell Lines Derived from Duchenne Muscular Dystrophy Patients

doi:

Figure Lengend Snippet: Up-regulation of RAMP mRNA expression in skeletal muscle after injury. A: Gastrocnemius muscle of mice was dissected 5 hours (lane 1) or 1 (lane 2), 2 (lane 3), 3 (lane 4), 4 (lane 5), 7 (lane 6), 10 (lane 7), or 14 (lane 8) days after injuring the muscle with a needle. Total RNA from each sample was extracted, reverse-transcribed, and amplified by 30 cycles of PCR using RAMP-specific primers. RT-PCR for GAPDH cDNA was performed to verify that equal amounts of template cDNA were used. B: Detection of RAMP mRNA in regenerating muscle fibers by in situ hybridization. Transverse cryosections of the tibialis anterior muscle of C57BL/10 mice harvested 6 days after crush injury were hybridized with digoxigenin-labeled anti-sense (a) or sense (b) cRNA probes for RAMP. Tibialis anterior muscle sections prepared from mdx mice after cardiotoxin injection were also hybridized with digoxigenin-labeled anti-sense (c) or sense (d) cRNA probes for RAMP. Specific signals for RAMP mRNA were detected as blue (a) or brown paints (c) in the centrally nucleated muscle fibers (arrows in a and c), but not in mature fibers (arrowheads in a and c). Scale bars, 50 μm.

Article Snippet: 23 By using cDNA derived from human skeletal muscle cells (Cambrex, Baltimore, MD) as the template, two polymerase chain reactions (PCRs) were independently performed with two sets of primers, namely, HD1 (5′-CTCGAGATGCTTTGGTGGGAAGAAGT-3′) and HD2(5′-TCTTTCAAGGGTATCCACAGTAATCTGCCTCTTC-3′) orHD3 (5′-ATTACTGTGGATACCCTTGAAAGACTCCAGGAAC-3′) and HD4 (5′-GCGGCCGCCTACATTGTGTCCTCTCTCAT-3′).

Techniques: Expressing, Reverse Transcription, Amplification, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Labeling, Injection

Journal:

Article Title: Cloning of cDNA Encoding a Regeneration-Associated Muscle Protease Whose Expression Is Attenuated in Cell Lines Derived from Duchenne Muscular Dystrophy Patients

doi:

Figure Lengend Snippet: Sequence and tissue distribution of RAMP. A: Sequences of human and mouse RAMP proteins. Predicted signal sequences are boxed. CUB and CCP/Sushi domains are indicated by a double line and a solid line, respectively. The trypsin-like serine protease domain is shown by a light gray box. B: Schematic representation of the structural motifs in RAMP. C: Predominant expression of RAMP mRNA in skeletal muscle and brain. Two μg of poly(A)+ RNA prepared from various organs of adult C57BL/6 mouse were electrophoresed, blotted, and hybridized with 32P-labeled mouse RAMP cDNA. The band size of the detected transcript is shown.

Article Snippet: 23 By using cDNA derived from human skeletal muscle cells (Cambrex, Baltimore, MD) as the template, two polymerase chain reactions (PCRs) were independently performed with two sets of primers, namely, HD1 (5′-CTCGAGATGCTTTGGTGGGAAGAAGT-3′) and HD2(5′-TCTTTCAAGGGTATCCACAGTAATCTGCCTCTTC-3′) orHD3 (5′-ATTACTGTGGATACCCTTGAAAGACTCCAGGAAC-3′) and HD4 (5′-GCGGCCGCCTACATTGTGTCCTCTCTCAT-3′).

Techniques: Sequencing, Expressing, Labeling

Journal:

Article Title: Cloning of cDNA Encoding a Regeneration-Associated Muscle Protease Whose Expression Is Attenuated in Cell Lines Derived from Duchenne Muscular Dystrophy Patients

doi:

Figure Lengend Snippet: Expression of RAMP mRNA in cell lines derived from human BMD and DMD patients. A: Myotube differentiation capacity of BMD-sm1, a representative cell line derived from a BMD patient’s skeletal muscle. The morphology is shown by phase contrast microscopy of the BMD-sm1 cell line cultured at 39.5°C (a) or 32.5°C (b). BMD-sm1 cells cultured at 39.5°C were immunostained with (d) or without (c) anti-desmin antibody. B: RT-PCR analysis of RAMP mRNA expression in nine human cell lines derived from skeletal muscle biopsies of patients. Total RNAs were extracted from the control (lane 1), two BMD (lanes 2 and 3), and six DMD (lanes 4 to 9) human muscle cell lines, reverse-transcribed, and amplified by 30 cycles of PCR using specific primers for human RAMP cDNA. Ethidium bromide-staining patterns of DNA bands are shown with β-tubulin, which shows that equal amounts of template cDNA were loaded. Scale bars, 150 μm.

Article Snippet: 23 By using cDNA derived from human skeletal muscle cells (Cambrex, Baltimore, MD) as the template, two polymerase chain reactions (PCRs) were independently performed with two sets of primers, namely, HD1 (5′-CTCGAGATGCTTTGGTGGGAAGAAGT-3′) and HD2(5′-TCTTTCAAGGGTATCCACAGTAATCTGCCTCTTC-3′) orHD3 (5′-ATTACTGTGGATACCCTTGAAAGACTCCAGGAAC-3′) and HD4 (5′-GCGGCCGCCTACATTGTGTCCTCTCTCAT-3′).

Techniques: Expressing, Derivative Assay, Microscopy, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription, Amplification, Staining