The mRNA levels of AMPK were measured as described earlier [32 (link)]. In brief, total RNA from skeletal muscle samples (~30 mg) was extracted with NucleoSpin® RNA/Protein (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. Analyses of the real-time quantitative PCR data were performed using the comparative threshold cycle [Ct] method as suggested by Applied Biosystems (User Bulletin #2). The following primers were used for AMPK:
Forward: 5’-GACTGGACATAAAGTTGCTGTGA-3’ 23,
Reverse: 5-’GGATTTTCCCGACCACGTC-3’ 19
The expression of mRNA of AMPK was normalised to beta-actin [32 (link)].
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Marton O., Koltai E., Takeda M., Koch L.G., Britton S.L., Davies K.J., Boldogh I, & Radak Z. (2014). Mitochondrial biogenesis-associated factors underlie the magnitude of response to aerobic endurance training in rats. Pflugers Archiv : European journal of physiology, 467(4), 779-788.
Beta actin Mrna Primers Protein Quantitative real time pcr analyses Skeletal muscle
Corresponding Organization :
Other organizations :
Semmelweis University, University of Michigan–Ann Arbor, University of Southern California, The University of Texas Medical Branch at Galveston
Beta-actin expression used for normalization of AMPK mRNA levels
controls
Positive control: None mentioned.
Negative control: None mentioned.
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