Mouse skeletal muscle C2C12 cells were initially plated on 15 cm plates in DMEM with 20% fetal bovine serum. At confluence, the cells were switched to low serum medium to initiate myogenic differentiation. For extraction of total RNA, cells were first rinsed in PBS and then lysed in Trizol reagent (Invitrogen catalog # 15596-026) either during exponential growth in high serum medium, or at 60 hrs, 5 days and 7 days after medium shift. Residual contaminating genomic DNA was removed from the total RNA fraction using Turbo DNA-free (Ambion catalog # AM1907M). mRNA was isolated from DNA-free total RNA using the Dynabeads mRNA Purification Kit (Invitrogen catalog # 610-06).
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C2C12 Myogenic Differentiation Protocol
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Other organizations : University of Maryland, College Park, California Institute of Technology, Washington University in St. Louis, University of California, Berkeley
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Variable analysis
independent variables
- Time after medium shift (60 hrs, 5 days, 7 days)
- Total RNA extraction
- MRNA isolation
- Cultured mouse skeletal muscle C2C12 cells
- Initial plating on 15 cm plates in DMEM with 20% fetal bovine serum
- Cells switched to low serum medium to initiate myogenic differentiation
- Cells rinsed in PBS before lysis in Trizol reagent
- Removal of contaminating genomic DNA from total RNA using Turbo DNA-free
- MRNA isolation from DNA-free total RNA using Dynabeads mRNA Purification Kit
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