Assessment of different preservation techniques for human skeletal muscle biopsy samples: A comparative method study on freeze‐drying, RNAlater, and RNAlater‐ICE

Human skeletal muscle comprises slow-twitch (type I) and fast-twitch (type II) fibers. Fiber type-specific analyses often require manual isolation of fibers, necessitating effective tissue preservation. While freeze-drying remains the standard, alternative preservation methods such as RNAlater and RNAlater-ICE are increasingly used. Besides their utility in preserving RNA, it needs to be determined whether RNAlater and RNAlater-ICE can be utilized for broader downstream biochemical analyses in skeletal muscle tissue. In this study, we compared freeze-drying to RNAlater and three RNAlater-ICE-based protocols. We observed substantial and consistent alterations in protein content, amino acid levels, and enzyme activity depending on the preservation method. Notably, all RNAlater-ICE protocols abolished citrate synthase activity, and branched-chain amino acid levels were markedly reduced in both RNAlater and RNAlater-ICE-treated samples relative to freeze-dried tissue. Total protein concentration was comparable between freeze-dried and RNAlater-preserved muscle, whereas RNAlater-ICE protocols yielded lower values. After centrifugation, supernatant protein concentration was higher in RNAlater-treated samples, but consistently lowest following RNAlater-ICE treatment. Our results demonstrate the importance of choosing an appropriate preservation method for skeletal muscle prior to downstream biochemical analysis and that care should be taken when using RNAlater and RNAlater-ICE for protein or amino acid analysis.